Pyrimido-pyrrolo-oxazine-dione compound inhibitors of the cystic fibrosis transmembrane conductance regulator protein and uses therefor

ABSTRACT

Provided herein are benzopyrimido-pyrrolo-oxazine-dione (BPO) compounds and pyrimido-pyrrolo-quinoxalinedione (PPQ) compounds, and compositions comprising these compounds, that inhibit cystic fibrosis transmembrane conductance regulator (CFTR) mediated ion transport and that are useful for treating diseases and disorders associated with aberrantly increased CFTR chloride channel activity, such as polycystic kidney disease and secretory diarrheas. The compounds and compositions comprising the compounds described herein may be used for inhibiting expansion or preventing formation of cysts in persons who have polycystic kidney disease.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a U.S. national stage application filed under 35U.S.C. §371 of International Patent Application PCT/US2012/039715,accorded an international filing date of May 25, 2012, which claimsbenefit under 35 U.S.C. §119(e) of U.S. Provisional Patent ApplicationSer. No. 61/491,151, filed May 27, 2011, which is incorporated herein byreference in its entirety.

STATEMENT OF GOVERNMENT INTEREST

This invention was made with government support under Grant NumbersDK86125, DK72517, HL73856, EP00415, DK35124, and EY13574 awarded by theNational Institutes of Health. The government has certain rights in thisinvention.

BACKGROUND

1. Technical Field

Therapeutics are needed for treating diseases and disorders related toaberrant cystic fibrosis transmembrane conductance regulator protein(CFTR)-mediated ion transport, such as polycystic kidney disease,increased intestinal fluid secretion, and secretory diarrhea. Smallmolecule compounds are described herein that are potent inhibitors ofCFTR activity and may be used for treating such diseases and disorders.

2. Description of the Related Art

The cystic fibrosis transmembrane conductance regulator protein (CFTR)is a cAMP-activated chloride (Cl⁻) channel expressed in epithelial cellsin mammalian airways, intestine, pancreas, and testis (see, e.g.,Sheppard et al., Physiol. Rev. 79:S23-45 (1999); Gadsby et al., Nature40:477-83 (2006)). Hormones, such as a β-adrenergic agonist, or a toxin,such as cholera toxin, lead to an increase in cAMP, activation ofcAMP-dependent protein kinase, and phosphorylation of the CFTR Cl⁻channel, which causes the channel to open. An increase in cell Ca²⁺ canalso activate different apical membrane channels. Phosphorylation byprotein kinase C can either open or shut Cl⁻ channels in the apicalmembrane. CFTR is predominantly located in epithelia where it provides apathway for the movement of Cl⁻ ions across the apical membrane and akey point at which to regulate the rate of transepithelial salt andwater transport.

CFTR chloride channel function is associated with a wide spectrum ofdisease, including cystic fibrosis (CF) and with polycystic kidneydisease, secretory diarrhea, and some forms of male infertility. Cysticfibrosis is a hereditary lethal disease caused by mutations in CFTR(see, e.g., Quinton, Physiol. Rev. 79:S3-S22 (1999); Boucher, Eur.Respir. J. 23:146-58 (2004)). Observations in human patients with CF andmouse models of CF indicate the functional importance of CFTR inintestinal and pancreatic fluid transport, as well as in male fertility(see, e.g., Grubb et al., Physiol. Rev. 79:S193-S214 (1999); Wong, Mol.Hum. Reprod. 4:107-110 (1997)). CFTR is also expressed in enterocytes inthe intestine and in cyst epithelium in polycystic kidney disease (see,e.g., O'Sullivan et al., Am. J. Kidney Dis. 32:976-983 (1998); Sullivanet al., Physiol. Rev. 78:1165-91 (1998); Strong et al., J. Clin. Invest.93:347-54 (1994); Mall et al., Gastroenterology 126:32-41 (2004);Hanaoka et al., Am. J. Physiol. 270:C389-C399 (1996); Kunzelmann et al.,Physiol. Rev. 82:245-289 (2002); Davidow et al., Kidney Int. 50:208-18(1996); Li et al., Kidney Int. 66:1926-38 (2004); Al-Awqati, J. Clin.Invest. 110:1599-1601 (2002); Thiagarajah et al., Curr. Opin. Pharmacol.3:594-99 (2003)).

Polycystic kidney disease (PKD) is one of the most common human geneticdiseases and a major cause of chronic renal insufficiency requiringdialysis and kidney transplantation (see, e.g., Torres et al., Lancet.369, 1287-301 (2007)). Cyst growth in PKD involves fluid secretion intothe cyst lumen coupled with epithelial cell hyperplasia. PKD ischaracterized by massive enlargement of fluid-filled cysts of renaltubular origin that compromise normal renal parenchyma and cause renalfailure (see, e.g., Arnaout, Annu. Rev. Med. 52: 93-123, 2001; Gabow N.Engl. J. Med. 329: 332-342, 1993; Harris et al., Mol. Genet. Metab. 81:75-85, 2004; Wilson N. Engl. J. Med. 350: 151-164, 2004; Sweeney et al.,Cell Tissue Res. 326: 671-685, 2006; Chapman J. Am. Soc. Nephrol. 18:1399-1407, 2007). Human autosomal dominant PKD (ADPKD) is caused bymutations in one of two genes, PKD1 and PKD2, encoding the interactingproteins polycystin-1 and polycystin-2, respectively (see, e.g., Wilson,supra; Qian et al., Cell 87: 979-987, 1996; Wu et al., Cell 93:177-88,1998; Watnick et al., Torres et al., Nat Med 10: 363-364, 2004 Nat.Genet. 25: 143-44 (2000)).

Cyst growth in autosomal dominant polycystic kidney disease (ADPKD)involves progressive fluid accumulation (see, e.g., Grantham et al.,Clin. J. Am. Soc. Nephrol. 1:148-57 (2006); Ye et al., N. Engl. J. Med329:310-13 (1993)). Fluid secretion into the cyst lumen requireschloride secretion by the cystic fibrosis transmembrane conductanceregulator (CFTR) protein, (see, e.g., Hanaoka et al., J. Am. Soc.Nephrol. 11:1179-87 (2000); Magenheimer et al., J. Am. Soc. Nephrol.17:3424-37 (2006)), a cAMP-regulated chloride channel, which, whenmutated, causes the genetic disease cystic fibrosis (see, e.g., Riordan,Annu. Rev. Biochem. 77:701-26 (2008)). CFTR is expressed strongly inepithelial cells lining cysts in ADPKD (see, e.g., Brill et al., Proc.Natl. Acad. Sci. USA 93:10206-11 (1996)). Cystic fibrosis (i.e.,CFTR-deficient) mice are resistant to cyst formation and CFTR inhibitorsblock cyst formation in cell/organ culture and in vivo models (see,e.g., Davidow et al., Kidney Int. 50:208-18 (1996); Li et al., KidneyInt. 66:1926-38 (2004)). In rare families affected with ADPKD and cysticfibrosis, individuals with both ADPKD and CF have less severe renaldisease than those with ADPKD only (see, e.g., Cotton et al., Am. J.Kidney Dis. 32:1081-83 (1998); O'Sullivan et al., Am. J. Kidney Dis.32:976-83 (1998); Xu et al., J. Nephrol. 19:529-34 (2006)).

Several CFTR inhibitors have been discovered, although many exhibit weakpotency and lack CFTR specificity. The oral hypoglycemic agentglibenclamide inhibits CFTR Cl⁻ conductance from the intracellular sideby an open channel blocking mechanism (see, e.g., Sheppard et al., J.Physiol., 503:333-346 (1997); Zhou et al., J. Gen. Physiol. 120:647-62(2002)) at high micromolar concentrations where it affects other Cl⁻ andcation channels (see, e.g., Edwards & Weston, 1993; Rabe et al., Br. J.Pharmacol. 110:1280-81 (1995); Schultz et al., Physiol. Rev.79:S109-S144 (1999)). Other non-selective anion transport inhibitors,including diphenylamine-2-carboxylate (DPC),5-nitro-2(3-phenylpropyl-amino)benzoate (NPPB), and flufenamic acid,also inhibit CFTR by occluding the pore at an intracellular site (see,e.g., Dawson et al., Physiol. Rev., 79:S47-S75 (1999); McCarty, J. Exp.Biol., 203:1947-62 (2000)).

High-affinity CFTR inhibitors also have clinical application in thetherapy of secretory diarrheas. Secretory diarrheas caused byenterotoxins, such as cholera and Travelers' diarrhea (enteropathogenicE. coli), require functional CFTR for primary chloride secretion intothe intestinal lumen, which secondarily drives sodium and watersecretion (see, e.g., Kunzelmann et al., Physiol. Rev. 82:245-89 (2002);Thiagarajah et al., Curr. Opin. Pharmacol. 3:594-9 (2003)). Cell cultureand animal models indicated that intestinal chloride secretion inenterotoxin-mediated secretory diarrheas occurs mainly through CFTR(see, e.g., Clarke et al., Science 257:1125-28 (1992); Gabriel et al.,Science 266:107-109 (1994); Kunzelmann and Mall, Physiol. Rev. 82:245-89(2002); Field, J. Clin. Invest. 111:931-43 (2003); and Thiagarajah etal., Gastroenterology 126:511-519 (2003)). Several classes of smallmolecule CFTR inhibitors have been described previously (see, e.g.,review by Verkman et al., Nat. Rev. Drug Discov. 8:153-71 (2009)).

Diarrheal disease in children is a global health concern: Approximatelyfour billion cases among children occur annually, resulting in at leasttwo million deaths. Travelers' diarrhea affects approximately 6 millionpeople per year. Antibiotics are routinely used to treat diarrhea;however, the antibiotics are ineffective for treating many pathogens,and the use of these drugs contributes to development of antibioticresistance in other pathogens. Oral replacement of fluid loss is alsoroutinely used to treat diarrhea, but is primarily palliative. Therapydirected at reducing intestinal fluid secretion (‘anti-secretorytherapy’) has the potential to overcome limitations of existingtherapies.

A need exists for CFTR inhibitors, particularly those that are safe,non-absorbable, highly potent, inexpensive, and chemically stable.

BRIEF SUMMARY

Briefly, provided herein are pyrimido-pyrrolo-oxazine-dione (BPO)compounds and certain pyrimido-pyrrolo-quinoxalinedione (PPQ) compounds,and compositions comprising such compounds, that inhibit cystic fibrosistransmembrane conductance regulator (CFTR) mediated ion transport. TheBPO and PPQ compounds described herein, are highly potent CFTRinhibitors, metabolically stable, and have desirable polarity and thushave excellent aqueous solubility. The compounds described herein areuseful for treating diseases and disorders associated with aberrantlyincreased CFTR chloride channel activity and thereby are useful fortreating diseases and disorders treatable by inhibiting CFTR-mediatedion transport. Methods are provided for inhibiting enlargement of kidneycysts or preventing or inhibiting the formation of cysts and therebytreating polycystic kidney disease by administering the compoundsdescribed herein. Methods are also provided for treating diseases anddisorders associated with aberrantly increased intestinal fluidsecretion, such as secretory diarrhea and Traveler's diarrhea, byadministering the compounds and compositions described herein.

Provided herein are compounds, compositions, methods and uses as setforth in the following embodiments.

Embodiment 1. A compound having the following structure (I):

as an isolated enantiomer or a racemic mixture of enantiomers, or as apharmaceutically acceptable salt, hydrate, solvate, N-oxide, or prodrugthereof, wherein:

m is 1, 2, 3, or 4;

n is 1, 2, 3, 4 or 5;

p is an integer from 0 to 4;

q is an integer from 1 to 4;

R¹ at each occurrence is the same or different and independently H,halo, haloalkyl, C₁₋₆ alkyl, —(CH₂)_(p)—C(O)—R^(4a), —S(O)₂R^(4a), —NO₂,or tetrazolyl;

R^(1a) at each occurrence is the same or different and independently H,halo, haloalkyl, C₁₋₆ alkyl, —(CH₂)_(p)—C(O)—R^(4a), —S(O)₂R^(4a), —NO₂,or tetrazolyl;

R^(2a) and R^(2b) are each the same or different and independently H orC₁-C₆ alkyl;

R^(4a) is —OR⁷, —NR⁷R⁸, —O(CH₂)_(q)—OC(O)R⁷, an amino acid residue, or apeptide;

R⁷ and R⁸ are each the same or different and independently H, C₁₋₂₀alkyl, a saccharide, an amino acid residue, or a peptide; and

Z is aryl or heteroaryl.

Embodiment 2. The compound of Embodiment 1, wherein R^(2a) and R^(2b)are each methyl, and Z is optionally substituted furanyl or optionallysubstituted thienyl, and the compound has the following structure (IA)or (IB), respectively:

as an isolated enantiomer or a racemic mixture of enantiomers, or as apharmaceutically acceptable salt, hydrate, solvate, N-oxide, or prodrugthereof,

wherein:

m is 1, 2, 3, or 4;

n is 1, 2, 3, 4 or 5;

p is an integer from 0 to 4;

q is an integer from 1 to 4;

R¹ at each occurrence is the same or different and independently H,halo, haloalkyl, C₁₋₆ alkyl, —(CH₂)_(p)—C(O)—R^(4a), —S(O)₂R^(4a), —NO₂,or tetrazolyl;

R^(1a) at each occurrence is the same or different and independently H,halo, haloalkyl, C₁₋₆ alkyl, —(CH₂)_(p)—C(O)—R^(4a), —S(O)₂R^(4a), —NO₂,or tetrazolyl;

R^(4a) is —OR⁷, —NR⁷R⁸, —O(CH₂)_(q)—OC(O)R⁷, an amino acid residue, or apeptide;

R⁵ is H, halo, or C₁₋₆ alkyl;

R⁶ is H, halo, C₁₋₆ alkyl, or C₁₋₆ haloalkyl; and

R⁷ and R⁸ are each the same or different and independently H, C₁₋₂₀alkyl, a saccharide, an amino acid residue, or a peptide.

Embodiment 3. The compound of Embodiment 1 or Embodiment 2, whereinR^(2a) and R^(2b) are each methyl, p is 0, R^(4a) is —OR⁷, Z isoptionally substituted furanyl, n is 1 and R¹ is meta to the linkingcarbon and the compound has the following structure (IA1):

wherein:

R¹ is H, halo, or C₁₋₆ alkyl;

R² and R³ are each the same or different and independently H, halo,—NO₂, C₁₋₆ alkyl, tetrazolyl, —S(O)₂OR⁷, or —C(═O)OR⁷;

R⁵ is H, halo, or C₁₋₆ alkyl;

R⁶ is halo, C₁₋₆ alkyl, or C₁₋₆ haloalkyl; and

R⁷ is H, C₁₋₆ alkyl, a saccharide, an amino acid residue, or a peptide.

Embodiment 4. The compound of Embodiment 1 or Embodiment 2, whereinR^(2a) and R^(2b) are each methyl, p is 0, R^(4a) is —OR⁷, Z isoptionally substituted thienyl, n is 1 and R¹ is meta to the linkingcarbon, and the compound has the following structure (IB1):

wherein:

R¹ is H, halo, or C₁₋₆ alkyl;

R² and R³ are each the same or different and independently H, halo,—NO₂, C₁₋₆ alkyl, tetrazolyl, —S(O)₂OR⁷, or —C(═O)OR⁷;

R⁵ is H, halo, or C₁₋₆ alkyl;

R⁶ is halo, C₁₋₆ alkyl, or C₁₋₆ haloalkyl; and

R⁷ is H, C₁₋₆ alkyl, a saccharide, an amino acid residue, or a peptide.

Embodiment 5. The compound of Embodiment 1, wherein R^(2a) and R^(2b)are each methyl, and Z is optionally substituted phenyl, and thecompound has the following structure (IC):

as an isolated enantiomer or a racemic mixture of enantiomers, or as apharmaceutically acceptable salt, hydrate, solvate, N-oxide, or prodrugthereof,

wherein:

m is 1, 2, 3, or 4;

n is 1, 2, 3, 4 or 5;

p is an integer from 0 to 4;

q is an integer from 1 to 4;

t is 1, 2, 3, 4 or 5;

R¹ at each occurrence is the same or different and independently H,halo, haloalkyl, C₁₋₆ alkyl, —(CH₂)_(p)—C(O)—R^(4a), —S(O)₂R^(4a), —NO₂,or tetrazolyl;

R^(1a) at each occurrence is the same or different and independently H,halo, haloalkyl, C₁₋₆ alkyl, —(CH₂)_(p)—C(O)—R^(4a), —S(O)₂R^(4a), —NO₂,or tetrazolyl;

R^(1b) at each occurrence is the same or different and independently H,halo, —OH, —NO₂, C₁₋₆ alkyl, C₂₋₆ alkenyl, or C₁₋₆ alkoxy;

R^(4a) is —OR⁷, —NR⁷R⁸, —O(CH₂)_(q)—OC(O)R⁷, an amino acid residue, or apeptide;

R⁷ and R⁸ are each the same or different and independently H, C₁₋₂₀alkyl, a saccharide, an amino acid residue, or a peptide.

Embodiment 6. The compound of Embodiment 1 or Embodiment 5, whereinR^(2a) and R^(2b) are each methyl, p is 0, R^(4a) is —OR⁷, n is 1 and R¹is meta to the linking carbon and the compound has the followingstructure:

wherein:

R¹ is H, halo, or C₁₋₆ alkyl;

R² and R³ are each the same or different and independently H, halo,—NO₂, C₁₋₆ alkyl, tetrazolyl, —S(O)₂OR⁷, or —C(═O)OR⁷;

t is 1, 2, 3, 4 or 5;

R^(1b) at each occurrence is the same or different and independently H,halo, —OH, —NO₂, C₁₋₆ alkoxy, or C₁₋₆ alkyl; and

R⁷ is H, C₁₋₆ alkyl, a saccharide, an amino acid residue, or a peptide.

Embodiment 7. A compound having the following structure (II):

as an isolated enantiomer or a racemic mixture of enantiomers, or as apharmaceutically acceptable salt, hydrate, solvate, N-oxide, or prodrugthereof,

wherein:

m is 1, 2, 3, or 4;

n is 1, 2, 3, 4 or 5;

p is an integer from 0 to 4;

q is an integer from 1 to 4;

X is O or S;

R¹ at each occurrence is the same or different and independently H,halo, haloalkyl, C₁₋₆ alkyl, —(CH₂)_(p)—C(O)—R^(4a), —S(O)₂R^(4a), —NO₂,or tetrazolyl;

R^(1a) at each occurrence is the same or different and independently H,halo, haloalkyl, C₁₋₆ alkyl, —(CH₂)_(p)—C(O)—R^(4a), —S(O)₂R^(4a), —NO₂,or tetrazolyl;

R^(2a) and R^(2b) are each the same or different and independently H orC₁₋₆ alkyl;

R^(4a) is —OR⁷, —NR⁷R⁸, —O(CH₂)_(q)—OC(O)R⁷, an amino acid residue, or apeptide;

R⁴ is H, —N(═O), C₁₋₆ alkyl, or haloalkyl;

R⁵ is H, halo, or C₁₋₆ alkyl;

R⁶ is halo, C₁₋₆ alkyl, or C₁₋₆ haloalkyl; and

R⁷ and R⁸ are each the same or different and independently H, C₁₋₂₀alkyl, a saccharide, an amino acid residue, or a peptide.

with the proviso that the following compounds are excluded:

(a)7,9-Dimethyl-11-(3-methylphenyl)-6-(5-methylfuran-2-yl)-5,6-dihydro-pyrimido[4′,5′-3,4]pyrrolo[1,2-a]quinoxaline-8,10-(7H,9H)-dione;

(b)7,9-Dimethyl-11-phenyl-6-(5-methylfuran-2-yl)-5,6-dihydro-pyrimido[4′,5′-3,4]pyrrolo[1,2-a]quinoxaline-8,10-(7H,9H)-dione;

(c)7,9-Dimethyl-11-(2-methylphenyl)-6-(5-methylfuran-2-yl)-5,6-dihydro-pyrimido[4′,5′-3,4]pyrrolo[1,2-a]quinoxaline-8,10-(7H,9H)-dione;

(d)2,3,7,9-Tetramethyl-11-phenyl-6-(5-methylfuran-2-yl)-5,6-dihydro-pyrimido[4′,5′-3,4]pyrrolo[1,2-a]quinoxaline-8,10-(7H,9H)-dione;

(e)2,3,7,9-Tetramethyl-11-(2-fluorophenyl)-6-(5-methylfuran-2-yl)-5,6-dihydro-pyrimido[4′,5′-3,4]pyrrolo[1,2-a]quinoxaline-8,10-(7H,9H)-dione;

(f)7,9-Dimethyl-11-(4-methylphenyl)-6-(5-methylfuran-2-yl)-5,6-dihydro-pyrimido[4′,5′-3,4]pyrrolo[1,2-a]quinoxaline-8,10-(7H,9H)-dione;and

(g)7,9-Dimethyl-11-(4-cholophenyl)-6-(5-methylfuran-2-yl)-5,6-dihydro-pyrimido[4′,5′-3,4]pyrrolo[1,2-a]quinoxaline-8,10-(7H,9H)-dione.

Embodiment 8. The compound of Embodiment 7, wherein R^(2a) and R^(2b)are each methyl, p is 0, R^(4a) is —OR⁷, n is 1 and R¹ is meta to thelinking carbon, and the compound has the following structure (IIA):

wherein:

X is O or S;

R¹ is H, halo, or C₁₋₃ alkyl;

R² is H, halo, —NO₂, or —C(═O)OR⁷;

R³ is H or —NO₂;

R⁴ is —N(═O), C₁₋₃ alkyl, or H;

R⁵ is H or C₁₋₃ alkyl;

R⁶ is C₁₋₆ alkyl, C₁₋₆ haloalkyl, or halo; and

R⁷ is H, C₁₋₆ alkyl, a saccharide, an amino acid residue, or a peptidewith the proviso that the following compounds are excluded:

7,9-Dimethyl-11-(3-methylphenyl)-6-(5-methylfuran-2-yl)-5,6-dihydro-pyrimido[4′,5′-3,4]pyrrolo[1,2-a]quinoxaline-8,10-(7H,9H)-dione;

7,9-Dimethyl-11-phenyl-6-(5-methylfuran-2-yl)-5,6-dihydro-pyrimido[4′,5′-3,4]pyrrolo[1,2-a]quinoxaline-8,10-(7H,9H)-dione;and

2,3,7,9-Tetramethyl-11-phenyl-6-(5-methylfuran-2-yl)-5,6-dihydro-pyrimido[4′,5′-3,4]pyrrolo[1,2-a]quinoxaline-8,10-(7H,9H)-dione.

Embodiment 9. The compound of Embodiment 1, wherein (a) each R¹ is H;(b) at least one R¹ is methyl or ethyl; (c) at least one R¹ is methyland each remaining R¹ is H; or (d) at least one R¹ is ethyl and eachremaining R¹ is H.

Embodiment 10. The compound of either Embodiment 1 or Embodiment 9,wherein m=2 and p=0 and (a) at least one of R^(1a) is halo, —NO₂, C₁₋₃alkyl, —C(O)—R^(4a); or (b) each of R^(1a) is H.

Embodiment 11. The compound of Embodiment 5, wherein (a) each R¹ is H;(b) at least one R¹ is methyl or ethyl; (c) at least one R¹ is methyland each remaining R¹ is H; or (d) at least one R¹ is ethyl and eachremaining R¹ is H.

Embodiment 12. The compound of Embodiment 5 or Embodiment 11, whereinm=2 and p=0 and (a) at least one of R^(1a) is halo, —NO₂, C₁₋₃ alkyl,—C(O)—R^(4a); or (b) each of R^(1a) is H.

Embodiment 13. The compound of Embodiment 6, wherein R¹ is H, methyl, orethyl.

Embodiment 14. The compound of Embodiment 6, wherein (a) R² is H, halo,—NO₂, or —C(═O)OR⁷, wherein R⁷ is H or C₁₋₆ alkyl; (b) R² is H, halo,—NO₂, or —C(═O)OR⁷, wherein R⁷ is H, —CH₃, —CH₂CH₃, or —CH₂CH₂CH₃; (c)R² is H, chloro, or —NO₂; (d) R² is —C(═O)OR⁷, wherein R⁷ is H, —CH₃, or—CH₂CH₃; or (e) R² is chloro.

Embodiment 15. The compound of Embodiment 6, wherein R³ is H or —NO₂.

Embodiment 16. The compound of any one Embodiments 5, 6, or 11-15,wherein (a) t=2 and each R^(1b) is the same or different andindependently H, —OH, halo, —NO₂, C₁₋₃ alkyl, or C₁₋₃ alkoxy; or (b) t=1and R^(1b) is —OH, halo, —NO₂, C₁₋₃ alkyl, or C₁₋₃ alkoxy.

Embodiment 17. The compound of any one Embodiments 5, 6, or 11-15,wherein (a) t=2 and each R^(1b) is the same or different andindependently H, —OH, chloro, fluoro, —NO₂, methyl, or methoxy; (b) t=1and R^(1b) is —OH, chloro, fluoro, —NO₂, methyl, or methoxy; or (c) eachR^(1b) is H.

Embodiment 18. The compound of Embodiment 8, wherein R⁴ is H, —N(═O), ormethyl.

Embodiment 19. The compound of any one of Embodiments 3, 4, 8, and 18,wherein R¹ is H, methyl, or ethyl.

Embodiment 20. The compound of any one of Embodiments 3, 4, 8, 18, and19 wherein (a) R² is H, halo, —NO₂, or —C(═O)OR⁷, wherein R⁷ is H orC₁-C₆ alkyl; (b) R² is H, halo, —NO₂, or —C(═O)OR⁷, wherein R⁷ is H,—CH₃, —CH₂CH₃, or —CH₂CH₂CH₃; (c) R² is H, chloro, or —NO₂; (d) R² is—C(═O)OR⁷, wherein R⁷ is H, —CH₃, or —CH₂CH₃; or (e) R² is chloro.

Embodiment 21. The compound of any one of Embodiments 3, 4, 8, 18-20,wherein R³ is H or —NO₂.

Embodiment 22. The compound of any one of Embodiments 3, 4, 8, and18-21, wherein R⁵ is H or methyl.

Embodiment 23. The compound of any one of Embodiments 3, 4, 8, and18-22, wherein R⁶ is C₁₋₃ alkyl, C₁₋₃ fluoroalkyl, chloro, bromo, iodo,or fluoro.

Embodiment 24. The compound of any one of Embodiments 3, 4, 8, and18-23, wherein R⁶ is methyl, ethyl, chloro, bromo, iodo, ortrifluoromethyl.

Embodiment 25. The compound of Embodiment 7, wherein R⁴ is H, —N(═O), ormethyl.

Embodiment 26. The compound of any one of Embodiments 2, 7, and 25,wherein (a) each R¹ is H; (b) at least one R¹ is methyl or ethyl; (c) atleast one R¹ is methyl and each remaining R¹ is H; or (d) at least oneR¹ is ethyl and each remaining R¹ is H.

Embodiment 27. The compound of any one of Embodiments 2, 7, 25, and 26,wherein m=2 and p=0 and (a) at least one of R^(1a) is halo, —NO₂, C₁₋₃alkyl, —C(O)—R^(4a); or (b) each of R^(1a) is H.

Embodiment 28. The compound of any one of Embodiments 2, 7, and 25-27,wherein (a) R⁵ is H or C₁₋₃ alkyl; or (b) R⁵ is H or methyl.

Embodiment 29. The compound of any one of Embodiments 2, 7, and 25-28,wherein (a) R⁶ is C₁-C₃ alkyl, C₁-C₃ fluoroalkyl, chloro, bromo, iodo,or fluoro; or (b) R⁶ is methyl, ethyl, trifluoromethyl, chloro, bromo,or iodo.

Embodiment 30. The compound of either Embodiment 7 or Embodiment 8,wherein X is O.

Embodiment 31. The compound of any one of Embodiments 1-3, wherein thecompound has any one of the following structures:

Embodiment 32. The compound of Embodiment 7 or Embodiment 8, wherein thecompound has any one of the following structures:

Embodiment 33. The compound of any one of Embodiments 1-32, wherein thecompound is an isolated enantiomer in R form.

Embodiment 34. The compound of Embodiment 33 wherein the compound is6R-(5-Bromofuran-2-yl)-7,9-dimethyl-8,10-dioxo-11-phenyl-7,8,9,10-tetrahydro-6H-benzo[b]pyrimido[4′,5′:3,4]pyrrolo[1,2-d][1,4]oxazine-2-carboxylicacid.

Embodiment 35. The compound of any one of Embodiments 1-32, wherein thecompound is an isolated enantiomer in S form.

Embodiment 36. A pharmaceutical composition comprising the compound ofany one of Embodiments 1-35 and a pharmaceutically suitable excipient.

Embodiment 37. A method for inhibiting cyst formation or inhibiting cystenlargement, said method comprising contacting (a) a cell that comprisesCFTR and (b) the pharmaceutical composition of Embodiment 36, underconditions and for a time sufficient that permit CFTR and the compoundto interact, wherein the compound inhibits CFTR-mediated ion transport.

Embodiment 38. A method for treating polycystic kidney diseasecomprising administering to a subject the composition of Embodiment 36.

Embodiment 39. The method of Embodiment 38, wherein polycystic kidneydisease is autosomal dominant polycystic kidney disease or autosomalrecessive polycystic kidney disease.

Embodiment 40. A method for treating a disease, condition, or disorderthat is treatable by inhibiting cystic fibrosis transmembraneconductance regulator (CFTR)-mediated ion transport, said methodcomprising administering to a subject the pharmaceutical composition ofEmbodiment 36, thereby inhibiting CFTR-mediated ion transport.

Embodiment 41. The method of Embodiment 40, wherein the disease,condition, or disorder is selected from polycystic kidney disease,aberrantly increased intestinal fluid secretion, and secretory diarrhea.

Embodiment 42. The method of Embodiment 41, wherein secretory diarrhea(a) is caused by an enteric pathogen; (b) is induced by an enterotoxin;or (c) is a sequelae of ulcerative colitis, irritable bowel syndrome(IBS), AIDS, chemotherapy, or an enteropathogenic infection.

Embodiment 43. Use of the compound of any one of Embodiments 1-35 fortreating a disease, condition, or disorder that is selected frompolycystic kidney disease, aberrantly increased intestinal fluidsecretion, and secretory diarrhea.

Embodiment 44. Use of the compound of any one of Embodiments 1-35 forthe manufacture of a medicament for treating a disease, condition, ordisorder that is selected from polycystic kidney disease, aberrantlyincreased intestinal fluid secretion, and secretory diarrhea.

Embodiment 45. The compound of any one of Embodiments 1-35 for use intreating a disease, condition, or disorder that is selected frompolycystic kidney disease, aberrantly increased intestinal fluidsecretion, and secretory diarrhea.

As used herein and in the appended claims, the singular forms “a,”“and,” and “the” include plural referents unless the context clearlydictates otherwise. Thus, for example, reference to “a compound” mayrefer to one or more compounds, or a plurality of such compounds, andreference to “a cell” or “the cell” includes reference to one or morecells and equivalents thereof (e.g., plurality of cells) known to thoseskilled in the art, and so forth. Similarly, reference to “acomposition” includes a plurality of such compositions, and refers toone or more compositions unless the context clearly dictates otherwise.When steps of a method are described or claimed, and the steps aredescribed as occurring in a particular order, the description of a firststep occurring (or being performed) “prior to” (i.e., before) a secondstep has the same meaning if rewritten to state that the second stepoccurs (or is performed) “subsequent” to the first step. The term“about” when referring to a number or a numerical range means that thenumber or numerical range referred to is an approximation withinexperimental variability (or within statistical experimental error), andthus the number or numerical range may vary between 1% and 15% of thestated number or numerical range. The term “comprising” (and relatedterms such as “comprise” or “comprises” or “having” or “including”) isnot intended to exclude that in other certain embodiments, for example,an embodiment of any composition of matter, composition, method, orprocess, or the like, described herein, may “consist of” or “consistessentially of” the described features.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the chemical structure of various CFTR inhibitors.

FIG. 2 presents a schematic of a representative synthesis procedure forpreparing PPQ compounds as described in Example 1 herein. Reagents: (a)NaOH, H₂O, dimethylsulfate, r.t. 3 days; (b) benzoyl chloride, ZnCl₂,chlorobenzene, reflux; (c) Br₂, CH₂Cl₂, cat. H₂O, reflux; (d)4-R²-1,2-phenylenediamine, EtOH, reflux; (e) R²═H, 4-R⁵-5-R⁶-furfural,1,2-dichloroethane, TsOH, reflux; X═S, 5-methylthiophene carbaldehyde,1,2-dichloroethane, cat. TsOH, reflux; R²═NO₂, 5-bromo-2-furaldehydeCHCl₃, cat. TFA, reflux; (f) R⁴═CH₃SO₂—, methanesulfonyl chloride, DCM,Et₃N; R⁴═CF₃CO—, TFA anhydride, DCM, Et₃N; R⁴═CH₃CO—, acetic anhydride,DMAP, 100° C.; R₄═NO, t-butyl nitrite, DCM; (g)N-methyl-1,2-phenylenediamine, EtOH, reflux; (h) 5-methylfurfural,1,2-dichloroethane, cat. TsOH, reflux.

FIG. 3 presents a schematic of a representative synthesis procedure forpreparing BPO compounds as described in Example 1 herein. Reagents: (a)R¹═H, benzoyl chloride, ZnCl₂, chlorobenzene, reflux; R¹=Me, m-tolylchloride, ZnCl₂, chlorobenzene, reflux; (b) Br₂, CH₂Cl₂, cat. H₂O,reflux; (c) 2-amino-3-R²-4-R³-phenol, EtOH, reflux; (d) 5-R⁶-furfural,cat. TFA, CHCl₃ or 1,2-dichloroethane, 150° C.; (e) KOH, THF, H₂O, HClworkup.

FIG. 4 shows chromatograms of purified BPO-27 enantiomers followingchiral HPLC separation. (A). Analytical chromatogram followingpreparation separation of approximately 1 g BPO-27. (B). Chromatogram offraction 1 analytical chromatograph showing the retention time (RT) and% area (A %) of peaks. (C). Analytical chromatogram of fraction #2.

FIG. 5 shows the X-ray crystal structure and absolute stereochemistry ofa compound of structure (I), BPO-25(S).

FIGS. 6A-6C illustrate metabolism of the compound, PPQ-102, in hepaticmicrosomes. FIG. 6A presents an LC/MS profile showing PPQ-102disappearance over 30 min during incubation with microsomes in thepresence of NADPH. FIG. 6B indicates the appearance of PPQ-102metabolites at +14 and +16 daltons. FIG. 6C presents a schematic ofpotential sites of PPQ-102 metabolism.

FIGS. 7A-7C present results from experiments performed to characterizePPQ analogs. FIG. 7A presents data from a fluorescence plate-readerassay of CFTR inhibition. FRT cells expressing human wildtype CFTR andiodide-sensing YFP fluorescent dye were incubated with test compound andCFTR agonists, and then subjected to an inwardly directed iodidegradient. FIG. 7A (top) shows representative data of the kinetics offluorescence decrease for PPQ-10, following iodide addition (causing YFPfluorescence quenching) in the absence of cAMP agonists and in thepresence of cAMP agonists at the indicated concentrations. FIG. 7A(bottom) presents a summary of concentration-inhibition data for theindicated compound (S.E. n=4). FIG. 7B presents data from short-circuitcurrent analysis of CFTR inhibition in CFTR-expressing FRT cells in thepresence of a transepithelial chloride gradient and basolateral membranepermeabilization. Where indicated, forskolin (20 μM) was added toactivate CFTR chloride conductance, following by indicatedconcentrations of PPQ-10. FIG. 7C (top) illustrates LC/MS analysisshowing disappearance of PPQ-10 and BPO-17 in hepatic microsomes in thepresence of NADPH. FIG. 7C (bottom) provides a summary of kinetics ofcompound disappearance (SEM, n=3). Data for PPQ-102 are shown forcomparison.

FIGS. 8A-8B illustrate data for PPQ CFTR inhibitors with high potency,metabolic stability and water solubility. FIG. 8A presents data fromshort-circuit current analysis that shows CFTR inhibition by compounds,BPO-21 and BPO-27. FIG. 8B illustrates the stability of the compounds inhepatic microsomes in the presence of NADPH.

FIGS. 9A and 9B demonstrate that compound BPO-27 reduced renalcytogenesis. FIG. 9A presents transmission light micrographs of E13.5embryonic kidneys cultured for the indicated days without or in thepresence of 100 μM 8-Br-cAMP, and with 0, 0.1, or 1 μM BPO-27. FIG. 9Bpresents a summary of percent cyst areas at 5 days in cultures (SEM,n=4-6, * P<0.001).

FIG. 10A-10B show CFTR inhibition activity of enantiomerically pureBPO-27 fractions. Short-circuit current was measured in FRT epithelialcells expressing human wildtype CFTR in presence of a transepithelialchloride gradient and following permeabilization of the basolateralmembrane. CFTR chloride conductance was stimulated by 10 μM forskolin.FIG. 10A shows BPO-27 fractions 2 and then 1 (each 100 nM) added whereindicated. FIG. 10B shows BPO-27 fraction 1 added at differentconcentrations. IC₅₀ was deduced as approximately 4 nM.

FIG. 11A-11C show in vitro metabolic stability and of BPO-27. FIG. 11A:In vitro metabolic stability of BPO-27 measured in hepatic microsomessupplemented with NADPH. (left) LC/MS chromatograms of non-metabolizedBPO-27 enantiomers at indicated times. (right) Percentagenon-metabolized compound remaining (S.E., n=4). FIG. 11B: In vivopharmacokinetics of BPO-27 in mice following bolus intraperitonealinjection of 300 mg/kg BPO-27. (left) Reference measurement. LC/MSchromatograms in which known amounts of BPO-27 was added to kidneyhomogenates and then extracted. Inset shows assay linearity. (right)LC/MS chromatograms of kidney homogenates at indicated times after bolusintraperitoneal injection. FIG. 11C: Concentration of BPO-27 measured inkidney, blood and urine following bolus intraperitoneal injection (S.E.,n=4).

DETAILED DESCRIPTION

Provided herein are pyrimido-pyrrolo-oxazine-dione (BPO) compounds thatinhibit activity of the cystic fibrosis transmembrane conductanceregulator (CFTR) chloride channel. Also provided herein are certainpyrimido-pyrrolo-quinoxalinedione (PPQ) compounds that inhibit CFTR. TheBPO and PPQ compounds described herein are capable of inhibitingCFTR-mediated ion transport (e.g., CFTR-mediated Cl⁻ transport) (i.e.,inhibiting CFTR conductance). The most potent BPO compounds havesubstantially improved metabolic stability (greater than 10-fold)compared with a previously characterized PPQ compound (called hereinPPQ-102; see, e.g., International Patent Application Publication No. WO2011/019737). These compounds disclosed herein also exhibit greaterpolarity and potency for inhibiting CFTR chloride conductance in vitro(˜10-fold) and renal cystogenesis ex vivo (>5-fold).

The compounds and compositions comprising these compounds are useful foradministering to a subject who has or who is at risk of developing adisease, disorder, or condition that is treatable (i.e., administrationof the compounds and compositions will provide a therapeutic orprophylactic benefit) by inhibiting CFTR-mediated ion transport. Thecompounds described herein may therefore be useful for treatingdiseases, disorders, and conditions including, for example, polycystickidney disease, secretory diarrhea, and other intestinal secretionconditions, diseases, and disorders characterized by aberrantlyincreased intestinal fluid secretion.

As noted above, autosomal dominant polycystic kidney disease (ADPKD) isa major health care burden and has a prevalence of 1 in 500 to 1000individuals (see, e.g., Torres et al., Lancet (2007), supra). NoFDA-approved drug is available at present that is capable of slowing theprogression of renal disease in PKD. Cyst expansion in ADPKD alsorequires cyst epithelial cell proliferation involving mTor signaling(see, e.g., Lieberthal et al., J. Am. Soc. Nephrol. 20:2493-502 (2009);Torres et al., Clin. J. Am. Soc. Nephrol. 5: 1312-29 (2010)), which isthe basis of several ‘anti-proliferative’ therapies under development(see, e.g., Belibi et al., Expert Opin. Investig. Drugs 19:315-28(2010); Torres, Clin. J. Am. Soc. Nephrol., 3:1212-18 (2008); Masoumi etal., Drugs 67:2495-2510 (2007); Patel et al., Curr Opin NephrolHypertens. 18:99-106 (2009); Torres, Adv. Chronic Kidney Dis. 17:190-204(2010)). “Anti-secretory” (e.g., CFTR inhibition) therapy is predictedto complement antiproliferative therapy or be effective alone inlife-long treatment of ADPKD. The BPO and PPQ compounds described hereinare CFTR inhibitors that may be useful as anti-secretory therapy forPKD. An alternative anti-secretory therapy, vasopressin V2 antagonism,is in clinical trials for PKD and is based on the concept that cysts inPKD often express V2 receptors, which when stimulated by antidiuretichormone, elevate cytoplasmic cAMP and activate both CFTR chlorideconductance and mTOR signaling (see, e.g., Belibi et al. (2010), supra;Torres, Clin. J. Am. Soc. Nephrol., 3:1212-18 (2008)). An alternative,V2 receptor-independent strategy has been suggested for reducing cAMPinvolving small-molecule phosphodiesterase activators, which were shownto resist growth in an in vitro PKD model (see, e.g., Tradtrantip etal., Mol. Pharmacol. 75:134-42 (2009)). Pure anti-proliferativetherapies, most based on the central role of mTOR signaling inproliferation of cyst-lining epithelial cells, are also in clinicaltrials, as well as renin-angiotensin inhibitors and statins (see, e.g.,Masoumi et al., Drugs (2007), supra; Patel et al., (2009), supra;Torres, (2010), supra).

Several classes of small-molecule CFTR inhibitors have been identifiedby high-throughput screening and characterized (see, e.g., Verkman etal., supra). Exemplary compounds are shown in FIG. 1.Thiazolidinone-class CFTR inhibitors such as CFTR_(inh)-172 act on thecytoplasmic side of the plasma membrane at a site near the CFTR pore toblock CFTR chloride conductance (see, e.g., Caci et al., Biochem.413:135-42 (2008); Ma et al., J. Clin. Invest. 110:1651-58 (2002);Taddei et al., FEBS Lett. 558:52-56 (2004); U.S. Pat. Nos. 7,235,573 and7,638,543). CFTR_(inh)-172 inhibits CFTR with IC₅₀ in the range of300-3000 nM, depending on cell type and membrane potential and has lowtoxicity and metabolic stability with primarily renal excretion (see,e.g., Ma et al., supra; Sonawane et al., J. Pharm. Sci. 94:134-43(2005)). The tetrazolo-substituted thiazolidinone Tetrazolo-172 (seeFIG. 1) had improved water solubility compared to CFTR_(inh)-172 (see,e.g., Sonawane et al., Bioorg. Med. Chem. 16:8187-95 (2008);International Patent Application Publication No. WO 09/120,803), andreduced cyst expansion in organ culture and mouse models of PKD (see,e.g., Yang et al., J. Am. Soc. Nephrol. 19:1300-10 (2008)). A secondclass of small-molecule CFTR inhibitors, the glycine hydrazides, such asGlyH-101 (see FIG. 1), block CFTR at an external site within the CFTRpore (see, e.g., Muanprasat et al., J. Gen. Physiol. 124:125-37 (2004);U.S. Pat. Nos. 7,414,037 and 7,888,332). Membrane-impermeant,non-absorbable conjugates of glycine hydrazides with polyethyleneglycols (see FIG. 1) (see, e.g., Sonawane et al., FASEB J. 20:130-32(2006); Sonawane et al., Chem. Biol. 15:718-28 (2008); U.S. Pat. Nos.7,414,037 and 7,888,332; U.S. Application Publication No.US2009/0253799; Int'l Patent Appl. Publication No. WO 09/146,144) andlectins (see, e.g., Sonawane et al., Gastroenterology 132:1234-44(2007); U.S. Application Publication No. 2008/0171793; Int'l PatentAppl. Publication No. WO 08/079,897) inhibit CFTR with an IC₅₀ of 50-100nM when added at the mucosal cell surface, and were effective in rodentmodels of secretory diarrheas such as cholera. An absorbable glycinehydrazide, phenyl-GlyH-101 (see FIG. 1), reduced cyst growth in PKD(see, e.g., Yang et al., 2008, supra; Int'l Patent Appl. Publication No.WO 09/120,803).

Additional small molecule screening yieldedpyrimido-pyrrolo-quinoxalinedione (PPQ) compounds, which were uncharged,and thus membrane-potential insensitive, CFTR inhibitors, and were verypotent CFTR inhibitors (Tradtrantip et al., J. Med. Chem. 52, 6447-55(2009); Int'l Patent Appl. Publication No. WO 2011/019737). Oneinhibitor, called PPQ-102 (7,9-dimethyl-11-phenyl-6-(5-methylfuran-2-yl)-5,6-dihydro-pyrimido-[4′,5′-3,4]pyrrolo[1,2-α]quinoxaline-8,10-(7H,9H)-dione,see FIG. 1) inhibited CFTR chloride current with IC₅₀˜90 nM, by amechanism involving stabilization of the channel closed-state. PPQ-102prevented cyst expansion and reduced the size of pre-formed cysts in anembryonic kidney organ culture model of PKD. However, PPQ-102 has poorermetabolic stability than desired, (precluding animal testing in certainanimal models), as well as low polarity and hence low aqueoussolubility.

Derivative compounds of PPQ compounds were prepared. The BPO compoundsdescribed herein exhibit significantly improved stability, watersolubility, and CFTR inhibition potency. The most potent compoundsdescribed herein have significantly improved potency more than 10-foldimproved metabolic stability, and much greater polarity and thusincreased aqueous solubility compared to PPQ-102. As described herein,the improved compounds were effective in preventing renal cyst expansionin a PKD model. By way of example with respect to CFTR inhibitoryactivity, the compound BPO-27 described herein has an IC₅₀ ofapproximately 8 nM compared with PPQ-102 having an IC₅₀ of 100 nM. ThePPQ and BPO compounds described herein may also be useful for treatingsecretory diarrheas and for investigating and elucidating CFTR-dependentcellular and physiological processes.

Pyrimido-Pyrrolo-Oxazine-Dione (BPO) andPyrimido-Pyrrolo-Quinoxalinedione (PPQ) Compounds

Provided herein are compounds that have the capability to inhibitCFTR-mediated ion transport. In one embodiment, a compound is providedthat has the following structure (I):

as an isolated enantiomer or a racemic mixture of enantiomers, or as apharmaceutically acceptable salt, hydrate, solvate, N-oxide, or prodrugthereof,

wherein:

m is 1, 2, 3, or 4;

n is 1, 2, 3, 4 or 5;

p is an integer from 0 to 4;

q is an integer from 1 to 4;

R¹ at each occurrence is the same or different and independently H,halo, haloalkyl, C₁₋₆ alkyl, —(CH₂)—C(O)—R^(4a), —S(O)₂R^(4a), —NO₂, ortetrazolyl;

R^(1a) at each occurrence is the same or different and independently H,halo, haloalkyl, C₁₋₆ alkyl, —(CH₂)_(p)—C(O)—R^(4a), —S(O)₂R^(4a), —NO₂,or tetrazolyl;

R^(2a) and R^(2b) are each the same or different and independently H orC₁₋₆ alkyl;

R^(4a) is —OR⁷, —NR⁷R⁸, —O(CH₂)_(q)—OC(O)R⁷, an amino acid residue, or apeptide;

R⁷ and R⁸ are each the same or different and independently H, C₁₋₂₀alkyl, a saccharide, an amino acid residue, or a peptide; and

Z is aryl or heteroaryl.

In more particular embodiments of the compound of structure (I), Z is a5-member, optionally substituted heteroaryl. In other more particularembodiments of the compound of structure (I), Z is optionallysubstituted phenyl. In certain particular embodiments, Z is optionallysubstituted furanyl, and in other certain embodiments, Z is optionallysubstituted thienyl.

In another particular embodiment of the compound of structure (I), n is1 or 2 and each R¹ is the same or different and independently H, halo,haloalkyl, C₁₋₆ alkyl, —(CH₂)_(p)—C(O)—R^(4a), —S(O)₂R^(4a), —NO₂, ortetrazolyl. In other specific embodiments, n is 1 and R¹ isunsubstituted C₁-C₆ alkyl or halo or H. In certain specific embodiments,R¹ is H at each occurrence. In other specific embodiments, n is 1 and R¹is unsubstituted C₁-C₆ alkyl. In a specific embodiment, n is 1 and R¹ isC₁-C₃ alkyl, which in more specific embodiments is unsubstituted. Inanother embodiment, n is 1 and R¹ is halo, which in certain embodimentsis chloro (Cl), fluoro (F), iodo (I), or bromo (Br). In one specificembodiment, n is 1, 2, or 3 and each R¹ is the same or different andindependently halo, methyl, or ethyl. In another particular embodiment,n is 1 and R¹ is methyl (—CH₃) or ethyl (—CH₂CH₃). In certain specificembodiments, at least one R¹ is methyl or ethyl and the remaining R¹ areeach H. In still another particular embodiment, n is 1 and R¹ is methyl.In still another specific embodiment, n is at least 1 and the at leastone R¹ is haloalkyl. In still other specific embodiments, n is 1 or 2and each R¹ is haloalkyl. In still another specific embodiment, n is 1and R¹ is C₁-C₆ haloalkyl. In more specific embodiments, n is 1 and R¹is C₁-C₃ haloalkyl. In another specific embodiment, n is 1 and R¹ isC₁-C₃ fluoroalkyl. In a more specific embodiment, the fluoroalkyl is atrifluoromethyl (—CF₃) or —CH₂CH₂CF₃. In another specific embodiment, nis 1 and R¹ is trifluoromethyl (—CF₃). In still another specificembodiment, n is 1 and R¹ is —CH₂CF₂CF₃. In other certain embodiments, nis 1 and R¹ is —NO₂. In still other certain embodiments, n is 1 and R¹is tetrazolyl (e.g., 5-tetrazolyl). In yet other certain embodiments, nis 1 and R¹ is —(CH₂)_(p)—C(O)—R^(4a). In yet other certain embodiments,n is 1 and R¹ is —S(O)₂R^(4a).

In still another specific embodiment, n is 1 or 2, and at least one ofR¹ is —S(O)₂R^(4a) or —(CH₂)_(p)—C(O)—R^(4a), and R^(4a) is —OR⁷,—NR⁷R⁸, or —O(CH₂)_(q)—OC(O)R⁷. In other certain embodiments, at leastone of R¹ is —S(O)₂R^(4a) or —(CH₂)_(p)—C(O)—R^(4a), and R^(4a) is anamino acid residue. In another certain embodiment, at least one of R¹ is—S(O)₂R^(4a) or —(CH₂)_(p)—C(O)—R^(4a), and R^(4a) is a peptide thatconsists of two (i.e., a dipeptide), three (i.e., a tripeptide), four,five, or six amino acid residues. In particular embodiments, R⁷ is H orC₁-C₆ alkyl. In yet another specific embodiment, R⁷ is C₁-C₃ alkyl,which in particular embodiments is unsubstituted, and which in otherparticular embodiments is methyl or ethyl. In still another specificembodiment, R⁷ is a saccharide. In particular embodiments, R⁸ is H orC₁-C₆ alkyl. In yet another specific embodiment, R⁸ is C₁-C₃ alkyl,which in particular embodiments is unsubstituted, and which in otherparticular embodiments is methyl or ethyl. In still another specificembodiment, R⁸ is a saccharide. In certain embodiments, R^(4a) is—NR⁷R⁸, and each of R⁷ and R⁸ is the same or different and independentlyH or C₁-C₆ alkyl. In certain embodiments, R^(4a) is —NR⁷R⁸, and each ofR⁷ and R⁸ is the same or different and independently H, methyl, orethyl. In other particular embodiments, R^(4a) is —OR⁷ or—O(CH₂)_(q)—OC(O)R⁷ and R⁷ is an amino acid residue. In still othercertain embodiments, R^(4a) is —OR⁷ or —O(CH₂)_(q)—OC(O)R⁷ and R⁷ is apeptide that consists of two (i.e., a dipeptide), three (i.e., atripeptide), four, five, or six amino acid residues. In still othercertain embodiments, R^(4a) is —NR⁷R⁸ and either R⁷ or R⁸ is an aminoacid residue. In still other certain embodiments, R^(4a) is —NR⁷R⁸ andeither R⁷ or R⁸ is a peptide that consists of two (i.e., a dipeptide),three (i.e., a tripeptide), four, five, or six amino acid residues.

In particular embodiments, when R¹ is —(CH₂)_(p)—C(O)—R^(4a), p is 0, 1,or 2. In one specific embodiment, p is 0, and R¹ is —C(O)—R^(4a). In yetother certain embodiments, n is 1 and R¹ is —(CH₂)_(p)—C(O)—R^(4a), andR^(4a) is —OR⁷. In certain particular embodiments, R^(4a) is −OR⁷, andR⁷ is hydrogen, C₁-C₂₀ alkyl, or a saccharide. In other certainembodiments, when R¹ is —(CH₂)_(p)—C(O)—R^(4a), and R^(4a) is —OR⁷, R⁷is hydrogen, methyl, ethyl. In other certain embodiments, when R¹ is—(CH₂)_(p)—C(O)—R^(4a), and R^(4a) is —OR⁷, R⁷ is a saccharide. In stillother particular embodiments, when R¹ is —(CH₂)_(p)—C(O)—R^(4a), andR^(4a) is —OR⁷, R⁷ is an amino acid residue or a peptide (i.e.,consisting of 2, 3, 4, 5, or six amino acids).

In another particular embodiment of the compound of structure (I), m is1 or 2. In another particular embodiment of the compound of structure(I), m is 1 or 2 and each R^(1a) is the same or different andindependently H, halo, haloalkyl, C₁-C₆ alkyl, —(CH₂)_(p)—C(O)—R^(4a),—S(O)₂R^(4a), —NO₂, or tetrazolyl. In certain specific embodiments,R^(1a) is H at each occurrence. In other particular embodiments, m is 1or 2 and at least one of R^(1a) is halo, which in particular embodimentshalo is I, Cl, or Br. In more specific embodiments, m is 1 or 2 and atleast one of R^(1a) is Cl. In yet another specific embodiment, m is 1and R^(1a) is Cl. In other specific embodiments, R^(1a) is unsubstitutedC₁-C₆ alkyl and m is 1 or 2. In a specific embodiment, m is 1 or 2 andat least one R^(1a) is C₁-C₃ alkyl, which in more specific embodimentsis unsubstituted. In another particular embodiment, m is 1 and R^(1a) ismethyl or ethyl. In still another particular embodiment, m is 1 andR^(1a) is methyl. In still other certain embodiments, m is 1 or 2, andat least one R^(1a) is tetrazolyl (e.g., 5-tetrazolyl). In still yetanother certain embodiments, m is 1 and R^(1a) is tetrazolyl. In anotherspecific embodiment, m is 1 or 2 and at least one of R^(1a) is —NO₂. Inanother specific embodiment, m is 1 and R^(1a) is —NO₂.

In more specific embodiments, m is 1 and R^(1a) is C₁-C₃ haloalkyl. Inanother specific embodiment, m is 1 and R^(1a) is C₁-C₃ fluoroalkyl. Ina more specific embodiment, the fluoroalkyl is a trifluoromethyl (—CF₃)or —CH₂CH₂CF₃. In another specific embodiment, m is 1 and R^(1a) istrifluoromethyl (—CF₃). In still another specific embodiment, m is 1 andR^(1a) is —CH₂CF₂CF₃.

In still another specific embodiment, m is 1 or 2, and at least one ofR^(1a) is —(CH₂)_(p)—C(O)_R^(4a), and R^(4a) is —OR⁷, —NR⁷R⁸, or—O(CH₂)_(q)—OC(O)R⁷. In particular embodiments, when R^(1a) is—(CH₂)_(p)—C(O)—R^(4a), p is 0, 1, or 2. In one particular embodiments,m is 2 and p is 0 and at least one of R^(1a) is halo, —NO₂, C₁-C₃ alkyl,—C(O)—R^(4a). In one specific embodiment, p is 0, and R¹ is—C(O)—R^(4a), wherein R^(4a) is —OR⁷ and R⁷ is H, —CH₃, —CH₂CH₃, or—CH₂CH₂CH₃. In certain particular embodiments, R^(4a) is —OR⁷, and R⁷ ishydrogen, C₁-C₂₀ alkyl, a saccharide, an amino acid residue, or apeptide (i.e., 2-6 amino acids). In other particular embodiments, R⁷ isH or C₁-C₆ alkyl. In yet another specific embodiment, R⁷ is C₁-C₃ alkyl,which in particular embodiments is unsubstituted, and which in otherparticular embodiments is methyl or ethyl. In still another specificembodiment, R⁷ is a saccharide (e.g., a monosaccharide, a disaccharide,a trisaccharide, or polysaccharide). In still other particularembodiments, R⁷ is an amino acid residue. In still another particularembodiment, R⁷ is a peptide.

In one embodiment when R^(1a) is —(CH₂)_(p)—C(O)—R^(4a), R^(4a) is —OR⁷,and R⁷ is hydrogen, C₁-C₂₀ alkyl, or a saccharide. In other particularembodiments, R⁷ is H or C₁-C₆ alkyl. In yet another specific embodiment,when R^(1a) is —(CH₂)_(p)—C(O)—R^(4a), and R^(4a) is —OR⁷, R⁷ is C₁-C₃alkyl, which in particular embodiments is unsubstituted, and which inother particular embodiments is methyl or ethyl. In still anotherspecific embodiment, when R^(1a) is —(CH₂)_(p)—C(O)—R^(4a) and R^(4a) is—OR⁷, R⁷ is a saccharide. In still another embodiment, R⁷ is an aminoacid residue. In still another particular embodiment, R⁷ is a peptide(i.e., 2-6 amino acids). In still more specific embodiments, when R^(1a)is —(CH₂)_(p)—C(O)—R^(4a), R^(4a) is —OR⁷, p is 0, and R⁷ is H, methyl,or ethyl.

In still one specific embodiment when R^(1a) is —(CH₂)_(p)—C(O)—R^(4a),R^(4a) is —NR⁷R⁸, wherein each of R⁷ and R⁸ is the same or different andindependently hydrogen, C₁-C₆ alkyl, a saccharide, an amino acid residueor a peptide (i.e., 2-6 amino acids). In certain embodiments, whenR^(4a) is —NR⁷R⁸, at least one or both of R⁷ and R⁸ is hydrogen. Inother certain embodiments, when R^(4a) is —NR⁷R⁸, at least one of R⁷ andR⁸ is hydrogen and the other is C₁-C₆ alkyl. In other certainembodiments, when R^(4a) is —NR⁷R⁸, at least one of R⁷ and R⁸ ishydrogen and the other is C₁-C₃ alkyl, which in particular embodimentsis unsubstituted, and which in other particular embodiments is methyl orethyl. In yet another embodiment when R^(4a) is —NR⁷R⁸, at least one ofR⁷ and R⁸ is hydrogen and the other is a saccharide. In yet anotherembodiment when R^(4a) is —NR⁷R⁸, at least one of R⁷ and R⁸ is hydrogenand the other is an amino acid residue or a peptide (i.e., 2-6 aminoacids).

In still another specific embodiment, when R^(1a) is—(CH₂)_(p)—C(O)—R^(4a), R^(4a) is —O(CH₂)_(q)—OC(O)R⁷ and R⁷ ishydrogen, C₁-C₂₀ alkyl, or a saccharide. In particular embodiments, p is1, 2, or 3; in other particular embodiments, q is 1, 2, or 3. In stillother specific embodiments, R⁷ is H or C₁-C₆ alkyl. In more specificembodiments, R⁷ is C₁-C₃ alkyl, which in particular embodiments isunsubstituted, and which in other particular embodiments R⁷ is methyl orethyl. In another embodiment, R⁷ is a saccharide. In still anotherembodiment, R⁷ is an amino acid residue. In still another particularembodiment, R⁷ is a peptide (i.e., 2-6 amino acids).

In a particular embodiment, at least one of R¹ or R^(1a) is a polargroup selected from —NO₂, tetrazolyl, —S(O)₂OR⁷, or —C(═O)OR⁷ (whereinR⁷ is hydrogen, C₁₋₂₀ alkyl, or a saccharide). In specific embodiments,R⁷ is H or C₁-C₆ alkyl. In more specific embodiments, R⁷ is C₁-C₃ alkyl,which in particular embodiments is unsubstituted, and which in otherparticular embodiments is methyl or ethyl. In another embodiment, R⁷ isa saccharide. In still another embodiment, R⁷ is an amino acid residue.In still another particular embodiment, R⁷ is a peptide (i.e., 2-6 aminoacids).

In other particular embodiments, R^(2a) or R^(2b) are each the same ordifferent and independently hydrogen or C₁-C₃ alkyl. In yet anotherparticular embodiments, R^(2a) or R^(2b) are each the same or differentand independently hydrogen, methyl, or ethyl. In certain embodiments,R^(2a) and R^(2b) are each H. In other certain embodiments, R^(2a) andR^(2b) are each methyl. In still other certain embodiments, R^(2a) andR^(2b) are each ethyl.

In further particular embodiments, the compound of structure (I) is anisolated R form.

In further particular embodiments, the compound of structure (I) is anisolated S form.

In one specific embodiment of the compound of structure (I), Z isoptionally substituted furanyl or optionally substituted thienyl, andthe compound has the following structure (Ia(i)) or I(b(i)),respectively.

as an isolated enantiomer or a racemic mixture of enantiomers, or as apharmaceutically acceptable salt, hydrate, solvate, N-oxide, or prodrugthereof, wherein each of R¹, R^(1a), R^(2a), and R^(2b) are defined asabove for a compound of structure (I), and wherein each of R⁵ and R⁶ aredefined as below for a compound of substructure (IA) or (IB).

In a more specific embodiment of the compound of structure (I), R^(2a)and R^(2b) are each methyl, and Z is optionally substituted furanyl oroptionally substituted thienyl, and the compound has the followingstructure (IA) or (IB), respectively.

as an isolated enantiomer or a racemic mixture of enantiomers, or as apharmaceutically acceptable salt, hydrate, solvate, N-oxide, or prodrugthereof, wherein:

m is 1, 2, 3, or 4;

n is 1, 2, 3, 4 or 5;

p is an integer from 0 to 4;

q is an integer from 1 to 4;

R¹ at each occurrence is the same or different and independently H,halo, haloalkyl, C₁-C₆ alkyl, —(CH₂)_(p)—C(O)—R^(4a), —S(O)₂R^(4a),—NO₂, or tetrazolyl;

R^(1a) at each occurrence is the same or different and independently H,halo, haloalkyl, C₁-C₆ alkyl, —(CH₂)_(p)—C(O)—R^(4a), —S(O)₂R^(4a),—NO₂, or tetrazolyl;

R^(4a) is —OR⁷, —NR⁷R⁸, —O(CH₂)_(q)—OC(O)R⁷, an amino acid residue, or apeptide;

R⁵ is H, halo, or C₁₋₆ alkyl;

R⁶ is H, halo, C₁₋₆ alkyl, or C₁-C₆ haloalkyl; and

R⁷ and R⁸ are each the same or different and independently H, C₁-C₂₀alkyl, a saccharide, an amino acid residue, or a peptide.

In a particular embodiment of the compound of structure (IA) or (IB), orstructure (Ia(i)) or (Ib(i)), n is 1 or 2 and each R¹ is the same ordifferent and independently H, halo, haloalkyl, C₁-C₆ alkyl,—(CH₂)_(p)—C(O)—R^(4a), —S(O)₂R^(4a), —NO₂, or tetrazolyl. In onespecific embodiment, n is 1, 2, or 3 and each R¹ is the same ordifferent and independently H, halo, methyl, or ethyl. In certainspecific embodiments, R¹ is H at each occurrence. In other specificembodiments, n is 1 and R¹ is unsubstituted C₁-C₆ alkyl or halo. Inother specific embodiments, n is 1 and R¹ is unsubstituted C₁-C₆ alkyl.In a specific embodiment, n is 1 and R¹ is C₁-C₃ alkyl, which in morespecific embodiments is unsubstituted. In another embodiment, n is 1 andR¹ is halo, which in certain embodiments is chloro, fluoro, iodo, orbromo. In another particular embodiment, n is 1 and R¹ is methyl orethyl. In still another particular embodiment, n is 1 and R¹ is methyl.In certain embodiments, at least one R¹ is methyl or ethyl and theremaining R¹ are each H. In still another specific embodiment n is atleast 1 and the at least one R¹ is haloalkyl. In still other specificembodiments, n is 1 or 2 and each R¹ is haloalkyl. In still anotherspecific embodiment, n is 1 and R¹ is C₁-C₆ haloalkyl. In more specificembodiments, n is 1 and R¹ is C₁-C₃ haloalkyl. In another specificembodiment, n is 1 and R¹ is C₁-C₃ fluoroalkyl. In a more specificembodiment, the fluoroalkyl is a trifluoromethyl (—CF₃) or —CH₂CH₂CF₃.In another specific embodiment, n is 1 and R¹ is trifluoromethyl (—CF₃).In still another specific embodiment, n is 1 and R¹ is —CH₂CF₂CF₃. Inother certain embodiments, n is 1 and R¹ is —NO₂. In still other certainembodiments, n is 1 and R¹ is tetrazolyl (e.g., 5-tetrazolyl). In yetother certain embodiments, n is 1 and R¹ is —(CH₂)_(p)—C(O)—R^(4a). Inyet other certain embodiments, n is 1 and R¹ is —S(O)₂R^(4a).

In still another specific embodiment, n is 1 or 2, and at least one ofR¹ is —S(O)₂R^(4a) or —(CH₂)_(p)—C(O)—R^(4a), and R^(4a) is —OR⁷,—NR⁷R⁸, or —O(CH₂)_(q)—OC(O)R⁷. In other certain embodiments, at leastone of R¹ is —S(O)₂R^(4a) or —(CH₂)_(p)—C(O)—R^(4a), and R^(4a) is anamino acid residue. In another certain embodiment, at least one of R¹ is—S(O)₂R^(4a) or —(CH₂)_(p)—C(O)—R^(4a), and R^(4a) is a peptide thatconsists of two (i.e., a dipeptide), three (i.e., a tripeptide), four,five, or six amino acid residues. In particular embodiments, R⁷ is H orC₁-C₆ alkyl. In yet another specific embodiment, R⁷ is C₁-C₃ alkyl,which in particular embodiments is unsubstituted, and which in otherparticular embodiments is methyl or ethyl. In still another specificembodiment, R⁷ is a saccharide. In particular embodiments, R⁸ is H orC₁-C₆ alkyl. In yet another specific embodiment, R⁸ is C₁-C₃ alkyl,which in particular embodiments is unsubstituted, and which in otherparticular embodiments is methyl or ethyl. In still another specificembodiment, R⁸ is a saccharide. In certain embodiments, R^(4a) is—NR⁷R⁸, and each of R⁷ and R⁸ is the same or different and independentlyH or C₁-C₆ alkyl. In certain embodiments, R^(4a) is —NR⁷R⁸, and each ofR⁷ and R⁸ is the same or different and independently H, methyl, orethyl. In other particular embodiments, R^(4a) is —OR⁷ or—O(CH₂)_(q)—OC(O)R⁷ and R⁷ is an amino acid residue. In still othercertain embodiments, R^(4a) is —OR⁷ or —O(CH₂)_(q)—OC(O)R⁷ and R⁷ is apeptide that consists of two (i.e., a dipeptide), three (i.e., atripeptide), four, five, or six amino acid residues. In still othercertain embodiments, R^(4a) is —NR⁷R⁸ and either R⁷ or R⁸ is an aminoacid residue. In still other certain embodiments, R^(4a) is —NR⁷R⁸ andeither R⁷ or R⁸ is a peptide that consists of two (i.e., a dipeptide),three (i.e., a tripeptide), four, five, or six amino acid residues.

In particular embodiments, when R¹ is —(CH₂)_(p)—C(O)—R^(4a), p is 0, 1,or 2. In yet other certain embodiments, n is 1 and R¹ is—(CH₂)_(p)—C(O)—R^(4a), and R^(4a) is —OR⁷. In certain particularembodiments, R^(4a) is —OR⁷, and R⁷ is hydrogen, C₁-C₂₀ alkyl, or asaccharide. In other certain embodiments, when R¹ is—(CH₂)_(p)—C(O)—R^(4a), and R^(4a) is —OR⁷, R⁷ is hydrogen, methyl,ethyl. In other certain embodiments, when R¹ is —(CH₂)_(p)—C(O)—R^(4a),and R^(4a) is —OR⁷, R⁷ is a saccharide. In still other particularembodiments, when R¹ is —(CH₂)_(p)—C(O)—R^(4a), and R^(4a) is —OR⁷, R⁷is an amino acid residue or a peptide (i.e., consisting of 2, 3, 4, 5,or six amino acids).

In another particular embodiment of the compound of structure (IA) or(IB), or structure (Ia(i)) or (Ib(i)), m is 1 or 2. In anotherparticular embodiment of the compound of structure (I), m is 1 or 2 andeach R^(1a) is the same or different and independently H, halo,haloalkyl, C₁-C₆ alkyl, —(CH₂)_(p)—C(O)—R^(4a), —S(O)₂R^(4a), —NO₂, ortetrazolyl. In certain specific embodiments, R^(1a) is H at eachoccurrence. In particular embodiments, m is 1 or 2 and at least one ofR^(1a) is halo, which in particular embodiments halo is I, Cl, or Br. Inmore specific embodiments, m is 1 or 2 and at least one of R^(1a) is Cl.In other specific embodiments, R^(1a) is unsubstituted C₁-C₆ alkyl and mis 1 or 2. In a specific embodiment, m is 1 or 2 and at least one R^(1a)is C₁-C₃ alkyl, which in more specific embodiments is unsubstituted. Inanother particular embodiment, m is 1 and R^(1a) is methyl or ethyl. Instill another particular embodiment, m is 1 and R^(1a) is methyl. Incertain embodiments, at least one R^(1a) is methyl or ethyl and theremaining R^(1a) are each H. In still other certain embodiments, m is 1or 2, and at least one R^(1a) is tetrazolyl (e.g., 5-tetrazolyl). Inanother specific embodiment, m is 1 or 2 and at least one of R^(1a) is—NO₂. In more specific embodiments, m is 1 and R^(1a) is C₁-C₃haloalkyl. In another specific embodiment, m is 1 and R^(1a) is C₁-C₃fluoroalkyl. In a more specific embodiment, the fluoroalkyl is atrifluoromethyl (—CF₃) or —CH₂CH₂CF₃. In another specific embodiment, mis 1 and R^(1a) is trifluoromethyl (—CF₃). In still another specificembodiment, m is 1 and R^(1a) is —CH₂CF₂CF₃.

In still another specific embodiment, m is 1 or 2, and at least one ofR^(1a) is —(CH₂)_(p)—C(O)—R^(4a), and R^(4a) is —OR⁷, —NR⁷R⁸, or—O(CH₂)_(q)—OC(O)R⁷. In particular embodiments, when R^(1a) is—(CH₂)_(p)—C(O)—R^(4a), p is 0, 1, or 2. In one particular embodiment, mis 2 and p is 0 and at least one of R^(1a) is halo, —NO₂, C₁-C₃ alkyl,—C(O)—R^(4a). In certain embodiments, R^(1a) is H at every occurrence.In one embodiment when R^(1a) is —(CH₂)_(p)—C(O)—R^(4a), R^(4a) is —OR⁷,and R⁷ is hydrogen, C₁-C₂₀ alkyl, a saccharide, an amino acid residue,or a peptide (i.e., 2-6 amino acids). In other particular embodiments,R⁷ is H or C₁-C₆ alkyl. In yet another specific embodiment, when R^(1a)is —(CH₂)_(p)—C(O)—R^(4a), and R^(4a) is —OR⁷, R⁷ is C₁-C₃ alkyl, whichin particular embodiments is unsubstituted, and which in otherparticular embodiments is methyl or ethyl. In still another specificembodiment, when R^(1a) is —(CH₂)_(p)—C(O)—R^(4a) and R^(4a) is —OR⁷, R⁷is a saccharide. In still more specific embodiments, when R^(1a) is—(CH₂)_(p)—C(O)—R^(4a), R^(4a) is —OR⁷, p is 0, and R⁷ is H, methyl, orethyl. In still other particular embodiments, R⁷ is an amino acidresidue. In still another particular embodiment, R⁷ is a peptide.

In still one specific embodiment when R^(1a) is —(CH₂)_(p)—C(O)—R^(4a),R^(4a) is —NR⁷R⁸, wherein each of R⁷ and R⁸ is the same or different andindependently hydrogen, C₁-C₆ alkyl, a saccharide, an amino acidresidue, or a peptide (i.e., 2-6 amino acids). In certain embodiments,when R^(4a) is —NR⁷R⁸, at least one or both of R⁷ and R⁸ is hydrogen. Inother certain embodiments, when R^(4a) is —NR⁷R⁸, at least one of R⁷ andR⁸ is hydrogen and the other is C₁-C₆ alkyl. In other certainembodiments, when R^(4a) is —NR⁷R⁸, at least one of R⁷ and R⁸ ishydrogen and the other is C₁-C₃ alkyl, which in particular embodimentsis unsubstituted, and which in other particular embodiments is methyl orethyl. In yet another embodiment when R^(4a) is —NR⁷R⁸, at least one ofR⁷ and R⁸ is hydrogen and the other is a saccharide. In yet anotherembodiment when R^(4a) is —NR⁷R⁸, at least one of R⁷ and R⁸ is hydrogenand the other is an amino acid residue or a peptide (i.e., 2-6 aminoacids).

In still another specific embodiment, when R^(1a) is—(CH₂)_(p)—C(O)_R^(4a), R^(4a) is —O(CH₂)_(q)—OC(O)R⁷ and R⁷ ishydrogen, C₁-C₂₀ alkyl, or a saccharide. In particular embodiments, p is1, 2, or 3; in other particular embodiments, q is 1, 2, or 3. In stillother specific embodiments, R⁷ is H or C₁-C₆ alkyl. In more specificembodiments, R⁷ is C₁-C₃ alkyl, which in particular embodiments isunsubstituted, and which in other particular embodiments is methyl orethyl. In another embodiment, R⁷ is a saccharide. In still anotherembodiment, R⁷ is an amino acid residue. In still another particularembodiment, R⁷ is a peptide (i.e., 2-6 amino acids).

In a particular embodiment, at least one of R¹ or R^(1a) is a polargroup selected from —NO₂, tetrazolyl, —S(O)₂OR⁷, or —C(═O)OR⁷ (whereinR⁷ is hydrogen, C₁₋₂₀ alkyl, a saccharide, an amino acid residue, or apeptide (i.e., 2-6 amino acids). In specific embodiments, R⁷ is H orC₁-C₆ alkyl. In more specific embodiments, R⁷ is C₁-C₃ alkyl, which inparticular embodiments is unsubstituted, and which in other particularembodiments is methyl or ethyl. In another embodiment, R⁷ is asaccharide. In still other particular embodiments, R⁷ is an amino acidresidue. In still another particular embodiment, R⁷ is a peptide.

In another specific embodiment of the compound of structure (IA) or(IB), R⁵ is H or C₁-C₃ alkyl. In one specific embodiment, R⁵ is H. Inanother specific embodiment, R⁵ is C₁-C₃ alkyl, which in particularembodiments is unsubstituted. In one embodiment, R⁵ is H or methyl. Instill another specific embodiment, R⁵ is methyl. In a more specificembodiment, each of R⁵ and R⁶ is methyl.

In yet another specific embodiment of the compound of structure (IA) or(IB), R⁶ is H, C₁-C₃ alkyl, C₁-C₃ fluoroalkyl, chloro, bromo, iodo, orfluoro. In another specific embodiment, R⁶ is methyl, ethyl,trifluoromethyl, chloro, bromo, or iodo. In another specific embodiment,R⁶ is C₁-C₆ alkyl. In another more specific embodiment, R⁶ is C₁-C₃alkyl. In more specific embodiments, R⁶ is unsubstituted C₁-C₆ alkyl. Inyet another specific embodiment, R⁶ is unsubstituted C₁-C₃ alkyl (i.e.,—CH₃, —CH₂CH₃, or —CH₂CH₂CH₃ (branched or straight chain)). In stillmore specific embodiments, R⁶ is methyl or ethyl. In still another morespecific embodiment, R⁶ is methyl. In yet another specific embodiment,R⁶ is C₁-C₆ haloalkyl. In more specific embodiments, R⁶ is C₁-C₃haloalkyl. In another specific embodiment, R⁶ is C₁-C₃ fluoroalkyl. In amore specific embodiment, the fluoroalkyl is a trifluoromethyl (—CF₃) or—CH₂CH₂CF₃. In another specific embodiment, R⁶ is trifluoromethyl(—CF₃). In still another specific embodiment, R⁶ is —CH₂CF₂CF₃. In yetanother particular embodiment, R⁶ is halo. In a more particularembodiment, R⁶ is chloro (Cl), bromo (Br), or iodo (I). In still anotherparticular embodiment, R⁶ is chloro. In yet another particularembodiment, R⁶ is bromo. In yet another particular embodiment, R⁶ isiodo.

In a particular embodiment, at least one of R¹ or R^(1a) is a polargroup selected from —NO₂, tetrazolyl, —S(O)₂OR⁷, or —C(═O)OR⁷ (whereinR⁷ is hydrogen, C₁-C₂₀ alkyl, a saccharide, an amino acid residue, or apeptide (i.e., 2-6 amino acids). In specific embodiments, R⁷ is H orC₁-C₆ alkyl. In more specific embodiments, R⁷ is C₁-C₃ alkyl, which inparticular embodiments is unsubstituted, and which in other particularembodiments is methyl or ethyl. In another embodiment, R⁷ is asaccharide. In still another embodiment, R⁷ is an amino acid residue. Instill another particular embodiment, R⁷ is a peptide (i.e., 2-6 aminoacids).

In further particular embodiments, the compound of structure (IA) or(IB) is an isolated R form.

In further particular embodiments, the compound of structure (IA) or(IB) is an isolated S form.

In another embodiment of the compound of structure (I) and (IA), whereinR^(2a) and R^(2b) are each methyl, p is 0, R^(4a) is —OR⁷, Z isoptionally substituted furanyl, n is 1, and R¹ is meta to the linkingcarbon and the compound has the following structure (IA1):

wherein:

R¹ is H, halo, or C₁-C₆ alkyl;

R² and R³ are each the same or different and independently H, halo,—NO₂, C₁-C₆ alkyl, tetrazolyl, —S(O)₂OR⁷, or —C(═O)OR⁷;

R⁵ is H, halo, or C₁-C₆ alkyl;

R⁶ is halo, C₁-C₆ alkyl, or C₁-C₆ haloalkyl; and

R⁷ is hydrogen, C₁-C₆ alkyl, a saccharide, an amino acid residue, or apeptide.

In a more specific embodiment of the compound of structure (IA1), R¹ isH, halo, methyl, or ethyl. In a more specific embodiment, R¹ is H. Inother specific embodiments, R¹ is unsubstituted C₁-C₆ alkyl or halo. Inother specific embodiments, R¹ is unsubstituted C₁-C₆ alkyl. In aspecific embodiment, R¹ is C₁-C₃ alkyl, which in more specificembodiments is unsubstituted. In another embodiment, R¹ is halo, whichin certain embodiments is chloro (Cl), fluoro (F), iodo (I), or bromo(Br). In another particular embodiment, R¹ is (—CH₃) or ethyl (—CH₂CH₃).In still another particular embodiment, R¹ is methyl.

In another particular embodiment of the compound of structure (IA1), R²is H, halo, —NO₂, or —C(═O)OR⁷, wherein R⁷ is H or C₁₋₆ alkyl. In aparticular embodiment, R² is H. In a more specific embodiment, R² is H,halo, —NO₂, or —C(═O)OR⁷, wherein R⁷ is H, —CH₃, —CH₂CH₃, or —CH₂CH₂CH₃.In another embodiment, R² is H, chloro, —NO₂, or —C(═O)OR⁷, wherein R⁷is H, —CH₃, or —CH₂CH₃. In another specific embodiment, R² is halo,which in particular embodiments is I, Cl, or Br. In more specificembodiments, R² is Cl. In another specific embodiment, R² is —NO₂. Inyet another specific embodiment, R² is tetrazolyl (e.g., 5-tetrazolyl).In still another specific embodiment, R² is —C(═O)OR⁷. In a moreparticular embodiment, when R² is —C(═O)OR⁷, R⁷ is hydrogen. In anothermore particular embodiment, R² is —C(═O)OR⁷, and R⁷ is C₁-C₆ alkyl,which in particular embodiments is unsubstituted. In yet anotherspecific embodiment, R² is —C(═O)OR⁷, and R⁷ is C₁-C₃ alkyl, which inparticular embodiments is unsubstituted. In still another specificembodiment, R² is —C(═O)OR⁷, and R⁷ is methyl or ethyl. In still otherparticular embodiments, R⁷ is an amino acid residue. In still anotherparticular embodiment, R⁷ is a peptide (i.e., 2-6 amino acids). In yetanother embodiment, R⁷ is a saccharide.

In yet another specific embodiment, R² is tetrazolyl (e.g.,5-tetrazolyl). In still another particular embodiment, R² is —S(O)₂OR⁷and R⁷ is H or C₁₋₆ alkyl. In more specific embodiments, R⁷ is C₁-C₃alkyl, which in particular embodiments is unsubstituted, and which inother particular embodiments is methyl or ethyl. In still anotherembodiment, R⁷ is a saccharide. In still other particular embodiments,R⁷ is an amino acid residue. In still another particular embodiment, R⁷is a peptide (i.e., 2-6 amino acids).

In another particular embodiment of the compound of structure (IA1), R³is H. In another specific embodiment, R³ is —NO₂.

In another specific embodiment of the compound of structure (IA1), R⁵ isH or C₁-C₃ alkyl. In one specific embodiment, R⁵ is H. In anotherspecific embodiment, R⁵ is C₁-C₃ alkyl, which in particular embodimentsis unsubstituted. In one embodiment, R⁵ is H or methyl. In still anotherspecific embodiment, R⁵ is methyl (—CH₃). In a more specific embodiment,each of R⁵ and R⁶ is methyl.

In yet another specific embodiment of the compound of structure (IA1),R⁶ is C₁-C₃ alkyl, C₁-C₃ fluoroalkyl, chloro, bromo, iodo, or fluoro. Inanother specific embodiment, R⁶ is methyl, ethyl, trifluoromethyl,chloro, bromo, or iodo. In another specific embodiment, R⁶ is C₁-C₆alkyl. In another more specific embodiment, R⁶ is C₁-C₃ alkyl. In morespecific embodiments, R⁶ is unsubstituted C₁-C₆ alkyl. In yet anotherspecific embodiment, R⁶ is unsubstituted C₁-C₃ alkyl (i.e., —CH₃,—CH₂CH₃, or —CH₂CH₂CH₃ (branched or straight chain)). In still morespecific embodiments, R⁶ is methyl (—CH₃) or ethyl (—CH₂CH₃). In stillanother more specific embodiment, R⁶ is methyl (—CH₃). In yet anotherspecific embodiment, R⁶ is C₁-C₆ haloalkyl. In more specificembodiments, R⁶ is C₁-C₃ haloalkyl. In another specific embodiment, R⁶is C₁-C₃ fluoroalkyl. In a more specific embodiment, the fluoroalkyl isa trifluoromethyl (—CF₃) or —CH₂CH₂CF₃. In another specific embodiment,R⁶ is trifluoromethyl (—CF₃). In still another specific embodiment, R⁶is —CH₂CF₂CF₃. In yet another particular embodiment, R⁶ is halo. In amore particular embodiment, R⁶ is chloro (Cl), bromo (Br), or iodo (I).In still another particular embodiment, R⁶ is chloro. In yet anotherparticular embodiment, R⁶ is bromo. In yet another particularembodiment, R⁶ is iodo.

In a particular embodiment, at least one of R² or R³ is a polar groupselected from —NO₂, tetrazolyl, —S(O)₂OR⁷, or —C(═O)OR⁷ (wherein R⁷ ishydrogen, C₁₋₂₀ alkyl, or a saccharide, an amino acid residue, or apeptide (i.e., 2-6 amino acids). In specific embodiments, R⁷ is H orC₁-C₆ alkyl. In more specific embodiments, R⁷ is C₁-C₃ alkyl, which inparticular embodiments is unsubstituted, and which in other particularembodiments is methyl or ethyl. In another embodiment, R⁷ is asaccharide. In still another embodiment, R⁷ is an amino acid residue. Instill another particular embodiment, R⁷ is a peptide (i.e., 2-6 aminoacids).

In further particular embodiments, the compound of structure (IA1) is anisolated R form.

In further particular embodiments, the compound of structure (IA1) is anisolated S form.

In certain specific embodiments, BPO compounds of structure (I),substructure (Ia(i)), substructure (IA), and substructure (IA1) are asfollows.

Compound Designation Compound Structure Compound Name BPO-16

6-(5-Bromofuran-2- yl)-7,9-dimethyl- 11-phenyl-6H- benzo[b]pyrimido[4′,5′- 3,4]pyrrolo[1,2- d]oxazine-8,10- (7H,9H)-dione BPO-17

7,9-Dimethyl-6-(5- iodofuran-2-yl)-11- phenyl-6H- benzo[b]pyrimido[4′,5′-3,4] pyrrolo[1,2- d]oxazine-8,10- (7H,9H)-dione BPO-18

7,9-Dimethyl-6-(5- methylfuran-2-yl)- 11-phenyl-6H- benzo[b]pyrimido[4′,5′-3,4] pyrrolo[1,2- d]oxazine-8,10- (7H,9H)-dione BPO-19

6-(5-Chlorofuran-2- yl)-7,9-dimethyl- 11-(m-tolyl)-6H- benzo[b]pyrimido[4′,5′-3,4] pyrrolo[1,2- d]oxazine-8,10- (7H,9H)-dione BPO-20

7,9-Dimethyl-6-(5- methylfuran-2-yl)- 11-(m-tolyl)-6H- benzo[b]pyrimido[4′,5′-3,4] pyrrolo[1,2- d]oxazine-8,10- (7H,9H)-dione BPO-21

6-(5-bromofuran-2- yl)-7,9-dimethyl-2- nitro-11-phenyl-6H-benzo[b]pyrimido [4′,5′-3,4] pyrrolo[1,2- d]oxazine-8,10-(7H,9H)-dione BPO-22

7,9-Dimethyl-6-(5- methylfuran-2-yl)- 2-nitro-11-phenyl-6H-benzo[b]pyrimido [4′,5′-3,4] pyrrolo[1,2- d]oxazine-8,10-(7H,9H)-dione BPO-24

2-Chloro-7,9- Dimethyl-6-(5- methylfuran-2-yl)- 3-nitro-11-phenyl-6H-benzo[b]pyrimido [4′,5′-3,4] pyrrolo[1,2- d]oxazine-8,10-(7H,9H)-dione BPO-25

Ethyl 6-(5- Bromofuran-2-yl)- 7,9-dimethyl-8,10- dioxo-11-phenyl-7,8,9,10-tetrahydro- 6H-benzo[b]pyrimido [4′,5′:3,4]pyrrolo[1,2-d][1,4]oxazine-2- carboxylate (R)-BPO-25

Ethyl 6R-(5- Bromofuran-2-yl)- 7,9-dimethyl-8,10- dioxo-11-phenyl-7,8,9,10-tetrahydro- 6H-benzo[b]pyrimido [4′,5′:3,4]pyrrolo[1,2-d][1,4]oxazine-2- carboxylate (S)-BPO-25

Ethyl 6S-(5- Bromofuran-2-yl)- 7,9-dimethyl-8,10- dioxo-11-phenyl-7,8,9,10-tetrahydro- 6H-benzo[b]pyrimido [4′,5′:3,4]pyrrolo[1,2-d][1,4]oxazine-2- carboxylate BPO-26

Ethyl 7,9- Dimethyl-8,10- dioxo-6-(5- methylfuran-2-yl)-11-phenyl-7,8,9,10- tetrahydro-6H- benzo[b]pyrimido [4′,5′:3,4]pyrrolo[1,2-d][1,4]oxazine-2- carboxylate BPO-27

6-(5-Bromofuran-2- yl)-7,9-dimethyl- 8,10-dioxo-11- phenyl-7,8,9,10-tetrahydro-6H- benzo[b]pyrimido [4′,5′:3,4]pyrrolo[1,2-d][1,4]oxazine-2- carboxylic acid (R)-BPO-27

6R-(5-Bromofuran- 2-yl)-7,9-dimethyl- 8,10-dioxo-11- phenyl-7,8,9,10-tetrahydro-6H- benzo[b]pyrimido [4′,5′:3,4]pyrrolo[1,2-d][1,4]oxazine-2- carboxylic acid (S)-BPO-27

6S-(5-Bromofuran- 2-yl)-7,9-dimethyl- 8,10-dioxo-11- phenyl-7,8,9,10-tetrahydro-6H- benzo[b]pyrimido [4′,5′:3,4]pyrrolo[1,2-d][1,4]oxazine-2- carboxylic acid

In another embodiment of the compound of structure (I) and (IB), R^(2a)and R^(2b) are each methyl, p is 0, R^(4a) is —OR⁷, Z is optionallysubstituted thienyl, and R¹ is meta to the linking carbon the compoundhas the following structure (IB1):

wherein:

R¹ is H, halo, or C₁₋₆ alkyl;

R² and R³ are each the same or different and independently H, halo,—NO₂, C₁-C₆ alkyl, tetrazolyl, —S(O)₂OR⁷, or —C(═O)OR⁷;

R⁵ is H, halo, or C₁-C₆ alkyl;

R⁶ is halo, C₁-C₆ alkyl, or C₁-C₆ haloalkyl; and

R⁷ is H, C₁-C₆ alkyl, a saccharide, an amino acid residue, or a peptide.

In a more specific embodiment of the compound of structure (IB 1), R¹ isH, halo, methyl, or ethyl. In a more specific embodiment, R¹ is H. Inother specific embodiments, R¹ is unsubstituted C₁-C₆ alkyl or halo. Inother specific embodiments, R¹ is unsubstituted C₁-C₆ alkyl. In aspecific embodiment, R¹ is C₁-C₃ alkyl, which in more specificembodiments is unsubstituted. In another embodiment, R¹ is halo, whichin certain embodiments is chloro, fluoro, iodo, or bromo. In anotherparticular embodiment, R¹ is methyl or ethyl. In still anotherparticular embodiment, R¹ is methyl.

In another particular embodiment of the compound of structure (IB 1), R²is H, halo, —NO₂, or —C(═O)OR⁷, wherein R⁷ is H or C₁-C₆ alkyl. In aparticular embodiment, R² is H. In a more specific embodiment, R² is H,halo, —NO₂, or —C(═O)OR⁷, wherein R⁷ is H, —CH₃, —CH₂CH₃, or —CH₂CH₂CH₃.In another embodiment, R² is H, chloro, —NO₂, or —C(═O)OR⁷, wherein R⁷is H, —CH₃, or —CH₂CH₃. In another specific embodiment, R² is halo,which in particular embodiments is I, Cl, or Br. In more specificembodiments, R² is Cl. In another specific embodiment, R² is —NO₂. Instill another specific embodiment, R² is —C(═O)OR⁷. In a more particularembodiment, R² is —C(═O)OR⁷, and R⁷ is hydrogen. In another moreparticular embodiment, R² is —C(═O)OR⁷, and R⁷ is C₁-C₆ alkyl, which inparticular embodiments is unsubstituted. In yet another specificembodiment, R² is —C(═O)OR⁷, and R⁷ is C₁-C₃ alkyl, which in particularembodiments is unsubstituted. In still another specific embodiment, R²is —C(═O)OR⁷, and R⁷ is methyl, or ethyl. In still another specificembodiment, R² is —C(═O)OR⁷, and R⁷ is H. In still another embodiment,R⁷ is an amino acid residue. In still another particular embodiment, R⁷is a peptide (i.e., 2-6 amino acids). In yet another embodiment, R⁷ is asaccharide.

In yet another specific embodiment, R² is tetrazolyl (e.g.,5-tetrazolyl). In still another particular embodiment, R² is —S(O)₂OR⁷and R⁷ is H or C₁-C₆ alkyl. In more specific embodiments, R⁷ is C₁-C₃alkyl, which in particular embodiments is unsubstituted, and which inother particular embodiments is methyl or ethyl. In still anotherembodiment, R⁷ is a saccharide. In still another embodiment, R⁷ is anamino acid residue. In still another particular embodiment, R⁷ is apeptide (i.e., 2-6 amino acids).

In another particular embodiment of the compound of structure (IB1), R³is H. In another specific embodiment, R³ is —NO₂.

In another specific embodiment of the compound of structure (IB1), R⁵ isH or C₁-C₃ alkyl. In one specific embodiment, R⁵ is H. In anotherspecific embodiment, R⁵ is C₁-C₃ alkyl, which in particular embodimentsis unsubstituted. In one embodiment, R⁵ is H or methyl. In still anotherspecific embodiment, R⁵ is methyl. In a more specific embodiment, eachof R⁵ and R⁶ is methyl.

In yet another specific embodiment of the compound of structure (IB 1),R⁶ is C₁-C₃ alkyl, C₁-C₃ fluoroalkyl, chloro, bromo, iodo, or fluoro. Inanother specific embodiment, R⁶ is methyl, ethyl, trifluoromethyl,chloro, bromo, or iodo. In another specific embodiment, R⁶ is C₁-C₆alkyl. In another more specific embodiment, R⁶ is C₁-C₃ alkyl. In morespecific embodiments, R⁶ is unsubstituted C₁-C₆ alkyl. In yet anotherspecific embodiment, R⁶ is unsubstituted C₁-C₃ alkyl (i.e., —CH₃,—CH₂CH₃, or —CH₂CH₂CH₃ (branched or straight chain)). In still morespecific embodiments, R⁶ is methyl or ethyl. In still another morespecific embodiment, R⁶ is methyl. In yet another specific embodiment,R⁶ is C₁-C₆ haloalkyl. In more specific embodiments, R⁶ is C₁-C₃haloalkyl. In another specific embodiment, R⁶ is C₁-C₃ fluoroalkyl. In amore specific embodiment, the fluoroalkyl is a trifluoromethyl (—CF₃) or—CH₂CH₂CF₃. In another specific embodiment, R⁶ is trifluoromethyl(—CF₃). In still another specific embodiment, R⁶ is —CH₂CF₂CF₃. In yetanother particular embodiment, R⁶ is halo. In a more particularembodiment, R⁶ is chloro, bromo, or iodo. In still another particularembodiment, R⁶ is chloro. In yet another particular embodiment, R⁶ isbromo. In yet another particular embodiment, R⁶ is iodo.

In certain particular embodiments of the compound of structure (IB1), R¹is H, methyl, or ethyl; R² and R³ are each the same or different andindependently H, halo, —NO₂, methyl, ethyl, or —C(═O)OR⁷ wherein R⁷ ishydrogen, methyl, or ethyl; R⁵ is H or methyl; and R⁶ is C₁-C₃ alkyl,C₁-C₃ fluoroalkyl, chloro, bromo, iodo, or fluoro.

In a particular embodiment, at least one of R² or R³ is a polar groupselected from —NO₂, tetrazolyl, —S(O)₂OR⁷, or —C(═O)OR⁷ (wherein R⁷ ishydrogen, C₁-C₂₀ alkyl, a saccharide, an amino acid residue, or apeptide (i.e., 2-6 amino acids).). In specific embodiments, R⁷ is H orC₁-C₆ alkyl. In more specific embodiments, R⁷ is C₁-C₃ alkyl, which inparticular embodiments is unsubstituted, and which in other particularembodiments is methyl or ethyl. In another embodiment, R⁷ is asaccharide. In still other particular embodiments, R⁷ is an amino acidresidue. In still another particular embodiment, R⁷ is a peptide.

In further particular embodiments, the compound of structure (IB1) is anisolated R form.

In further particular embodiments, the compound of structure (IB1) is anisolated S form.

In a more specific embodiment of the compound of structure (I), R^(2a)and R^(2b) are each methyl, and Z is optionally substituted phenyl, andthe compound has the following structure (IC).

as an isolated enantiomer or a racemic mixture of enantiomers, or as apharmaceutically acceptable salt, hydrate, solvate, N-oxide, or prodrugthereof,

wherein:

m is 1, 2, 3, or 4;

n is 1, 2, 3, 4 or 5;

p is an integer from 0 to 4;

q is an integer from 1 to 4;

t is 1, 2, 3, 4 or 5;

R¹ at each occurrence is the same or different and independently H,halo, haloalkyl, C₁-C₆ alkyl, —(CH₂)_(p)—C(O)—R^(4a), —S(O)₂R^(4a),—NO₂, or tetrazolyl;

R^(1a) at each occurrence is the same or different and independently H,halo, haloalkyl, C₁-C₆ alkyl, —(CH₂)_(p)—C(O)—R^(4a), —S(O)₂R^(4a),—NO₂, or tetrazolyl;

R^(1b) at each occurrence is the same or different and independently H,halo, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₁-C₆ alkoxy, —NO₂, or —OH;

R^(4a) is −OR⁷, —NR⁷R⁸, —O(CH₂)_(q)—OC(O)R⁷, an amino acid residue, or apeptide;

R⁷ and R⁸ are each the same or different and independently hydrogen,C₁-C₂₀ alkyl, a saccharide, an amino acid residue, or a peptide.

In another particular embodiment of the compound of substructure (IC), nis 1 or 2 and each R¹ is the same or different and independently H,halo, haloalkyl, C₁-C₆ alkyl, —(CH₂)_(p)—C(O)—R^(4a), —S(O)₂R^(4a),—NO₂, or tetrazolyl. In certain specific embodiments, R¹ is H at eachoccurrence. In one specific embodiment, n is 1, 2, or 3 and each R¹ isthe same or different and independently H, halo, methyl, or ethyl. Inother specific embodiments, n is 1 and R¹ is unsubstituted C₁-C₆ alkylor halo. In other specific embodiments, R¹ is unsubstituted C₁-C₆ alkyland n is 1. In a specific embodiment, n is 1 and R¹ is C₁-C₃ alkyl,which in more specific embodiments is unsubstituted. In anotherembodiment, n is 1 and R¹ is halo, which in certain embodiments ischloro, fluoro, iodo, or bromo. In another particular embodiment, n is 1and R¹ is methyl or ethyl. In still another particular embodiment, n is1 and R¹ is methyl. In certain embodiments, at least one R¹ is methyl orethyl and the remaining R¹ are each H. In still another specificembodiment n is at least 1 and the at least one R¹ is haloalkyl. Instill other specific embodiments, n is 1 or 2 and each R¹ is haloalkyl.In still another specific embodiment, n is 1 and R¹ is C₁-C₆ haloalkyl.In more specific embodiments, n is 1 and R¹ is C₁-C₃ haloalkyl. Inanother specific embodiment, n is 1 and R¹ is C₁-C₃ fluoroalkyl. In amore specific embodiment, the fluoroalkyl is a trifluoromethyl (—CF₃) or—CH₂CH₂CF₃. In another specific embodiment, n is 1 and R¹ istrifluoromethyl (—CF₃). In still another specific embodiment, n is 1 andR¹ is —CH₂CF₂CF₃. In other certain embodiments, n is 1 and R¹ is —NO₂.In still other certain embodiments, n is 1 and R¹ is tetrazolyl (e.g.,5-tetrazolyl). In yet other certain embodiments, n is 1 and R¹ is—(CH₂)_(p)—C(O)—R^(4a). In yet other certain embodiments, n is 1 and R¹is —S(O)₂R^(4a).

In still another specific embodiment, n is 1 or 2, and at least one ofR¹ is —S(O)₂R^(4a) or —(CH₂)_(p)—C(O)—R^(4a), and R^(4a) is —OR⁷,—NR⁷R⁸, or —O(CH₂)_(q)—OC(O)R⁷. In other certain embodiments, at leastone of R¹ is —S(O)₂R^(4a) or —(CH₂)_(p)—C(O)—R^(4a), and R^(4a) is anamino acid residue. In another certain embodiment, at least one of R¹ is—S(O)₂R^(4a) or —(CH₂)_(p)—C(O)—R^(4a), and R^(4a) is a peptide thatconsists of two (i.e., a dipeptide), three (i.e., a tripeptide), four,five, or six amino acid residues. In particular embodiments, R⁷ is H orC₁-C₆ alkyl. In yet another specific embodiment, R⁷ is C₁-C₃ alkyl,which in particular embodiments is unsubstituted, and which in otherparticular embodiments is methyl or ethyl. In still another specificembodiment, R⁷ is a saccharide. In particular embodiments, R⁸ is H orC₁-C₆ alkyl. In yet another specific embodiment, R⁸ is C₁-C₃ alkyl,which in particular embodiments is unsubstituted, and which in otherparticular embodiments is methyl or ethyl. In still another specificembodiment, R⁸ is a saccharide. In certain embodiments, R^(4a) is—NR⁷R⁸, and each of R⁷ and R⁸ is the same or different and independentlyH or C₁-C₆ alkyl. In certain embodiments, R^(4a) is —NR⁷R⁸, and each ofR⁷ and R⁸ is the same or different and independently H, methyl, orethyl. In other particular embodiments, R^(4a) is —OR⁷ or—O(CH₂)_(q)—OC(O)R⁷ and R⁷ is an amino acid residue. In still othercertain embodiments, R^(4a) is —OR⁷ or —O(CH₂)_(q)—OC(O)R⁷ and R⁷ is apeptide that consists of two (i.e., a dipeptide), three (i.e., atripeptide), four, five, or six amino acid residues. In still othercertain embodiments, R^(4a) is —NR⁷R⁸ and either R⁷ or R⁸ is an aminoacid residue. In still other certain embodiments, R^(4a) is —NR⁷R⁸ andeither R⁷ or R⁸ is a peptide that consists of two (i.e., a dipeptide),three (i.e., a tripeptide), four, five, or six amino acid residues.

In particular embodiments, when R¹ is —(CH₂)_(p)—C(O)—R^(4a), p is 0, 1,or 2. In yet other certain embodiments, n is 1 and R¹ is—(CH₂)_(p)—C(O)—R^(4a), and R^(4a) is —OR⁷. In certain particularembodiments, R^(4a) is —OR⁷, and R⁷ is hydrogen, C₁-C₂₀ alkyl, or asaccharide. In other certain embodiments, when R¹ is—(CH₂)_(p)—C(O)—R^(4a), and R^(4a) is —OR⁷, R⁷ is hydrogen, methyl,ethyl. In other certain embodiments, when R¹ is —(CH₂)_(p)—C(O)—R^(4a),and R^(4a) is —OR⁷, R⁷ is a saccharide. In still other particularembodiments, when R¹ is —(CH₂)_(p)—C(O)—R^(4a), and R^(4a) is —OR⁷, R⁷is an amino acid residue or a peptide (i.e., consisting of 2, 3, 4, 5,or six amino acids).

In another particular embodiment of the compound of structure (IC), m is1 or 2. In another particular embodiment of the compound of structure(I), m is 1 or 2 and each R^(1a) is the same or different andindependently H, halo, haloalkyl, C₁-C₆ alkyl, —(CH₂)_(p)—C(O)—R^(4a),—S(O)₂R^(4a), —NO₂, or tetrazolyl. In one particular embodiment, m is 2and p is 0 and at least one of R^(1a) is halo, —NO₂, C₁-C₃ alkyl,—C(O)—R^(4a). In certain embodiments, R^(1a) is H at every occurrence.In particular embodiments, m is 1 or 2 and at least one of R^(1a) ishalo, which in particular embodiments halo is I, Cl, or Br. In morespecific embodiments, m is 1 or 2 and at least one of R^(h) is Cl. Inother specific embodiments, R^(1a) is unsubstituted C₁-C₆ alkyl and m is1 or 2. In a specific embodiment, m is 1 or 2 and at least one R^(1a) isC₁-C₃ alkyl, which in more specific embodiments is unsubstituted. Inanother particular embodiment, m is 1 and R^(1a) is methyl or ethyl. Instill another particular embodiment, m is 1 and R^(1a) is methyl. Instill other certain embodiments, m is 1 or 2, and at least one R^(1a) istetrazolyl (e.g., 5-tetrazolyl). In another specific embodiment, m is 1or 2 and at least one of R^(1a) is —NO₂. In more specific embodiments, mis 1 and R^(1a) is C₁-C₃ haloalkyl. In another specific embodiment, m is1 and R^(1a) is C₁-C₃ fluoroalkyl. In a more specific embodiment, thefluoroalkyl is a trifluoromethyl (—CF₃) or —CH₂CH₂CF₃. In anotherspecific embodiment, m is 1 and R^(1a) is trifluoromethyl (—CF₃). Instill another specific embodiment, m is 1 and R^(1a) is —CH₂CF₂CF₃.

In still another specific embodiment, m is 1 or 2, and at least one ofR^(1a) is —(CH₂)_(p)—C(O)—R^(4a), and R^(4a) is —OR⁷, —NR⁷R⁸, or—O(CH₂)_(q)—OC(O)R⁷. In particular embodiments, when R^(1a) is—(CH₂)_(p)—C(O)—R^(4a), p is 0, 1, or 2. In one embodiment when R^(1a)is —(CH₂)_(p)—C(O)_R^(4a), R^(4a) is —OR⁷, and R⁷ is hydrogen, C₁-C₂₀alkyl, a saccharide, an amino acid residue, or a peptide (i.e., 2-6amino acids). In other particular embodiments, R⁷ is H or C₁-C₆ alkyl.In yet another specific embodiment, when R^(1a) is—(CH₂)_(p)—C(O)—R^(4a), and R^(4a) is —OR⁷, R⁷ is C₁-C₃ alkyl, which inparticular embodiments is unsubstituted, and which in other particularembodiments R⁷ is methyl or ethyl. In still another specific embodiment,when R^(1a) is —(CH₂)_(p)—C(O)—R^(4a) and R^(4a) is —OR⁷, R⁷ is asaccharide. In still other particular embodiments, R⁷ is an amino acidresidue. In still another particular embodiment, R⁷ is a peptide. Instill more specific embodiments, when R^(1a) is —(CH₂)_(p)—C(O)—R^(4a),p is 0, R^(4a) is —OR⁷, and R⁷ is H, methyl, or ethyl.

In still one specific embodiment when R^(1a) is —(CH₂)_(p)—C(O)—R^(4a),R^(4a) is —NR⁷R⁸, wherein each of R⁷ and R⁸ is the same or different andindependently hydrogen, C₁-C₆ alkyl, or a saccharide. In certainembodiments, when R^(4a) is —NR⁷R⁸, at least one or both of R⁷ and R⁸ ishydrogen. In other certain embodiments, when R^(4a) is —NR⁷R⁸, at leastone of R⁷ and R⁸ is hydrogen and the other is C₁-C₆ alkyl. In othercertain embodiments, when R^(4a) is —NR⁷R⁸, at least one of R⁷ and R⁸ ishydrogen and the other is C₁-C₃ alkyl, which in particular embodimentsis unsubstituted, and which in other particular embodiments is methyl orethyl. In yet another embodiment when R^(4a) is —NR⁷R⁸, at least one ofR⁷ and R⁸ is hydrogen and the other is a saccharide. In yet anotherembodiment when R^(4a) is —NR⁷R⁸, at least one of R⁷ and R⁸ is hydrogenand the other is an amino acid residue or a peptide (i.e., 2-6 aminoacids).

In still another specific embodiment, when R^(1a) is—(CH₂)_(p)—C(O)—R^(4a), R^(4a) is —O(CH₂)_(q)—OC(O)R⁷ and R⁷ ishydrogen, C₁-C₂₀ alkyl, or a saccharide. In particular embodiments, p is1, 2, or 3; in other particular embodiments, q is 1, 2, or 3. In stillother specific embodiments, R⁷ is H or C₁-C₆ alkyl. In more specificembodiments, R⁷ is C₁-C₃ alkyl, which in particular embodiments isunsubstituted, and which in other particular embodiments is methyl orethyl. In another embodiment, R⁷ is a saccharide. In still anotherembodiment, R⁷ is an amino acid residue. In still another particularembodiment, R⁷ is a peptide (i.e., 2-6 amino acids).

In a particular embodiment, at least one of R¹ or R^(1a) is a polargroup selected from —NO₂, tetrazolyl, —S(O)₂OR⁷, or —C(═O)OR⁷ (whereinR⁷ is hydrogen, C₁-C₂₀ alkyl, or a saccharide). In specific embodiments,R⁷ is H or C₁-C₆ alkyl. In more specific embodiments, R⁷ is C₁-C₃ alkyl,which in particular embodiments is unsubstituted, and which in otherparticular embodiments is methyl or ethyl. In another embodiment, R⁷ isa saccharide. In still another embodiment, R⁷ is an amino acid residue.In still another particular embodiment, R⁷ is a peptide (i.e., 2-6 aminoacids).

In certain embodiments of the compound of substructure (IC), t is 1, 2,or 3. In other particular embodiments, when t is 1, 2, or 3, each R^(1b)is the same or different and independently H, halo, C₁-C₆ alkyl, C₂-C₆alkenyl, C₁-C₆ alkoxy, nitro (—NO₂), or —OH. In other specificembodiments, t=2 and each R^(1b) is the same or different andindependently H, —OH, halo, —NO₂, C₁-C₃ alkyl, or C₁-C₃ alkoxy. Inanother specific embodiment, t=1 and R^(1b) is —OH, halo, —NO₂, C₁-C₃alkyl, or C₁-C₃ alkoxy. In still another specific embodiment, t=1 andR^(1b) is —OH, chloro, fluoro, —NO₂, methyl, or methoxy. In yet morespecific embodiments, t=2 and each R^(1b) is the same or different andindependently H, —OH, chloro, fluoro, —NO₂, methyl, or methoxy. In otherspecific embodiments, R^(1b) is H at each occurrence. In specificembodiments, R^(1b) is halo selected from bromo, chloro, iodo, andfluoro. In more specific embodiments, R^(1b) is chloro. In otherparticular embodiments, R^(1b) is fluoro. In other specific embodiments,R^(1b) is C₁-C₆ alkyl. In certain embodiments, R^(1b) is C₂-C₆ alkenyl,and in other particular embodiments, R^(1b) is C₁-C₆ alkoxy. In stillother specific embodiments, R^(1b) is C₁-C₃ alkyl (e.g., methyl, ethyl,propyl). In other specific embodiments, R^(1b) is C₁-C₃ alkoxy. In moreparticular embodiments, R^(1b) is methoxy or ethoxy. In still otherembodiments, R^(1b) is hydroxyl (—OH), and in yet another specificembodiment, R^(1b) is —NO₂. In other particular embodiments, t is 1 or2. In still other embodiments, t is 2 and each R^(1b) is the same ordifferent and selected from methyl, chloro, fluoro, methoxy, nitro, andhydroxyl. In a particular embodiment, at least one of R¹ or R^(1a) is apolar group selected from —NO₂, tetrazolyl, —S(O)₂OR⁷, and —C(═O)OR⁷(wherein R⁷ is hydrogen, C₁₋₂₀ alkyl, or a saccharide). In specificembodiments, R⁷ is H or C₁-C₆ alkyl. In more specific embodiments, R⁷ isC₁-C₃ alkyl, which in particular embodiments is unsubstituted, and whichin other particular embodiments is methyl or ethyl. In anotherembodiment, R⁷ is a saccharide. In still another embodiment, R⁷ is anamino acid residue. In still another particular embodiment, R⁷ is apeptide (i.e., 2-6 amino acids).

In further particular embodiments, the compound of structure (IC) is anisolated R form.

In further particular embodiments, the compound of structure (IC) is anisolated S form.

In another embodiment of the compound of structure (I) and (IC), whereinR^(2a) and R^(2b) are each methyl, p is 0, R^(4a) is —OR⁷, n is 1, andR′ is meta to the linking carbon and the compound has the followingstructure:

wherein:

R¹ is H, halo, or C₁₋₆ alkyl;

R² and R³ are each the same or different and independently H, halo,—NO₂, C₁-C₆ alkyl, tetrazolyl, —S(O)₂OR⁷, or —C(═O)OR⁷;

t is 1, 2, 3, 4 or 5;

R^(1b) at each occurrence is the same or different and independently H,halo, —NO₂, C₁₋₆ alkoxy, C₂-C₆ alkenyl or C₁₋₆ alkyl;

R⁷ is hydrogen, C₁-C₆ alkyl, a saccharide, an amino acid residue, or apeptide.

In a more specific embodiment of the compound of structure (IC1), R¹ isH, halo, methyl, or ethyl. In a more specific embodiment, R¹ is H. Inother specific embodiments, R¹ is unsubstituted C₁-C₆ alkyl or halo. Inother specific embodiments, R¹ is unsubstituted C₁-C₆ alkyl. In aspecific embodiment, R¹ is C₁-C₃ alkyl, which in more specificembodiments is unsubstituted. In another embodiment, R¹ is halo, whichin certain embodiments is chloro, fluoro, iodo, or bromo. In anotherparticular embodiment, R¹ is methyl or ethyl. In still anotherparticular embodiment, R¹ is methyl.

In another particular embodiment of the compound of structure (IC1), R²is H, halo, —NO₂, or —C(═O)OR⁷, wherein R⁷ is H or C₁₋₆ alkyl. In aparticular embodiment, R² is H. In a more specific embodiment, R² is H,halo, —NO₂, or —C(═O)OR⁷, wherein R⁷ is H, —CH₃, —CH₂CH₃, or —CH₂CH₂CH₃.In another embodiment, R² is H, chloro, —NO₂, or —C(═O)OR⁷, wherein R⁷is H, —CH₃, or —CH₂CH₃. In another specific embodiment, R² is halo,which in particular embodiments is I, Cl, or Br. In more specificembodiments, R² is Cl. In another specific embodiment, R² is —NO₂. Instill another specific embodiment, R² is —C(═O)OR⁷. In a more particularembodiment, R² is —C(═O)OR⁷, and R⁷ is hydrogen. In another moreparticular embodiment, R² is —C(═O)OR⁷, and R⁷ is C₁-C₆ alkyl, which inparticular embodiments is unsubstituted. In yet another specificembodiment, R² is —C(═O)OR⁷, and R⁷ is C₁-C₃ alkyl, which in particularembodiments is unsubstituted. In still another specific embodiment, R²is —C(═O)OR⁷, and R⁷ is methyl or ethyl).

In another particular embodiment of the compound of structure (IC1), R³is H. In another specific embodiment, R³ is —NO₂.

In certain embodiments of the compound of substructure (IC1), t is 1 or2. In other particular embodiments, when t is 1 or 2, each R^(1b) is thesame or different and independently H, halo, C₁-C₆ alkyl, C₂-C₆ alkenyl,C₁-C₆ alkoxy, nitro (—NO₂), or —OH. In other specific embodiments, t=2and each R^(1b) is the same or different and independently H, —OH, halo,—NO₂, C₁-C₃ alkyl, or C₁-C₃ alkoxy. In another specific embodiment, t=1and R^(1b) is —OH, halo, —NO₂, C₁-C₃ alkyl, or C₁-C₃ alkoxy. In stillanother specific embodiment, t=1 and R^(1b) is —OH, chloro, fluoro,—NO₂, methyl, or methoxy. In yet more specific embodiments, t=2 and eachR^(1b) is the same or different and independently H, —OH, chloro,fluoro, —NO₂, methyl, or methoxy. In other specific embodiments, R^(1b)is H at each occurrence. In specific embodiments, at least one R^(1b) ishalo selected from bromo, chloro, iodo, and fluoro. In more specificembodiments, at least one R^(1b) is chloro. In other particularembodiments, at least one R^(1b) is fluoro. In other specificembodiments, at least one R^(1b) is C₁-C₆ alkyl. In certain embodiments,at least one R^(1b) is C₁-C₆ alkoxy. In still other specificembodiments, the at least one R^(1b) is C₁-C₃ alkyl (e.g., methyl,ethyl, propyl). In other specific embodiments, at least one R^(1b) isC₁-C₃ alkoxy. In more particular embodiments, at least one R^(1b) ismethoxy or ethoxy. In still other embodiments, at least one R^(1b) ishydroxyl (—OH), and in yet another specific embodiment, at least oneR^(1b) is —NO₂. In certain embodiments, t is 2 and each R^(1b) is thesame or different and selected from methyl, chloro, fluoro, methoxy,nitro, and hydroxyl. In certain particular embodiments, t is 1 andR^(1b) is selected from methyl, chloro, fluoro, methoxy, nitro, andhydroxyl.

In further particular embodiments, the compound of structure (IC1) is anisolated R form.

In further particular embodiments, the compound of structure (IC1) is anisolated S form.

In a further embodiment, a compound is provided that has the followingstructure (II):

as an isolated enantiomer or a racemic mixture of enantiomers, or as apharmaceutically acceptable salt, hydrate, solvate, N-oxide, or prodrugthereof,

wherein:

m is 1, 2, 3, or 4;

n is 1, 2, 3, 4 or 5;

p is an integer from 0 to 4;

q is an integer from 1 to 4;

X is O or S;

R¹ at each occurrence is the same or different and independently H,halo, haloalkyl, C₁₋₆ alkyl, —(CH₂)_(p)—C(O)—R^(4a), —S(O)₂R^(4a), —NO₂,or tetrazolyl;

R^(1a) at each occurrence is the same or different and independently H,halo, haloalkyl, C₁-C₆ alkyl, —(CH₂)_(p)—C(O)—R^(4a), —S(O)₂R^(4a),—NO₂, or tetrazolyl;

R^(2a) and R^(2b) are each the same or different and independently H orC₁₋₆ alkyl;

R^(4a) is —OR⁷, —NR⁷R⁸, —O(CH₂)_(q)—OC(O)R⁷, an amino acid residue, or apeptide;

R⁴ is hydrogen, —N(═O), C₁₋₆ alkyl, or haloalkyl;

R⁵ is H, halo, or C₁₋₆ alkyl;

R⁶ is halo, C₁₋₆ alkyl, or C₁₋₆ haloalkyl; and

R⁷ and R⁸ are each the same or different and independently hydrogen,C₁₋₂₀ alkyl, a saccharide, an amino acid residue, or a peptide.

with the proviso that the following compounds are excluded:

(a)7,9-Dimethyl-11-(3-methylphenyl)-6-(5-methylfuran-2-yl)-5,6-dihydro-pyrimido[4′,5′-3,4]pyrrolo[1,2-a]quinoxaline-8,10-(7H,9H)-dione;

(b)7,9-Dimethyl-11-phenyl-6-(5-methylfuran-2-yl)-5,6-dihydro-pyrimido[4′,5′-3,4]pyrrolo[1,2-a]quinoxaline-8,10-(7H,9H)-dione;

(c)7,9-Dimethyl-11-(2-methylphenyl)-6-(5-methylfuran-2-yl)-5,6-dihydro-pyrimido[4′,5′-3,4]pyrrolo[1,2-a]quinoxaline-8,10-(7H,9H)-dione;

(d)2,3,7,9-Tetramethyl-11-phenyl-6-(5-methylfuran-2-yl)-5,6-dihydro-pyrimido[4′,5′-3,4]pyrrolo[1,2-a]quinoxaline-8,10-(7H,9H)-dione;

(e)2,3,7,9-Tetramethyl-11-(2-fluorophenyl)-6-(5-methylfuran-2-yl)-5,6-dihydro-pyrimido[4′,5′-3,4]pyrrolo[1,2-a]quinoxaline-8,10-(7H,9H)-dione;

(f)7,9-Dimethyl-11-(4-methylphenyl)-6-(5-methylfuran-2-yl)-5,6-dihydro-pyrimido[4′,5′-3,4]pyrrolo[1,2-a]quinoxaline-8,10-(7H,9H)-dione;and

(g)7,9-Dimethyl-11-(4-cholophenyl)-6-(5-methylfuran-2-yl)-5,6-dihydro-pyrimido[4′,5′-3,4]pyrrolo[1,2-a]quinoxaline-8,10-(7H,9H)-dione.

The chemical structures of the excluded compounds are set forth in TableA below.

TABLE A PPQ-101

(a) 7,9-Dimethyl-11-(3- methylphenyl)-6-(5-methylfuran-2-yl)-5,6-dihydro-pyrimido[4′,5′- 3,4]pyrrolo[1,2-a]quinoxaline-8,10-(7H,9H)-dione PPQ-102

(b) 7,9-Dimethyl-11-phenyl-6-(5- methylfuran-2-yl)-5,6-dihydro-pyrimido[4′,5′-3,4]pyrrolo[1,2- a]quinoxaline-8,10-(7H,9H)-dione PPQ-103

(c) 7,9-Dimethyl-11-(2- methylphenyl)-6-(5-methylfuran-2-yl)-5,6-dihydro-pyrimido[4′,5′- 3,4]pyrrolo[1,2-a]quinoxaline-8,10-(7H,9H)-dione PPQ-104

(d) 2,3,7,9-Tetramethyl-11-phenyl-6- (5-methylfuran-2-yl)-5,6-dihydro-pyrimido[4′,5′-3,4]pyrrolo[1,2- a]quinoxaline-8,10-(7H,9H)-dione PPQ-105

(e) 2,3,7,9-Tetramethyl-11-(2- fluorophenyl)-6-(5-methylfuran-2-yl)-5,6-dihydro-pyrimido[4′,5′- 3,4]pyrrolo[1,2-a]quinoxaline-8,10-(7H,9H)-dione PPQ-110

(f) 7,9-Dimethyl-11-(4- methylphenyl)-6-(5-methylfuran-2-yl)-5,6-dihydro-pyrimido[4′,5′- 3,4]pyrrolo[1,2-a]quinoxaline-8,10-(7H,9H)-dione PPQ-111

(g) 7,9-Dimethyl-11-(4- cholophenyl)-6-(5-methylfuran-2-yl)-5,6-dihydro-pyrimido[4′,5′- 3,4]pyrrolo[1,2-a]quinoxaline-8,10-(7H,9H)-dione

In certain embodiments of the compound of structure (II), X is O. Inother certain embodiments of the compound of structure (II), X is S.

In a particular embodiment of the compound of structure (II), n is 1 or2 and each R¹ is the same or different and independently H, halo,haloalkyl, C₁-C₆ alkyl, —(CH₂)_(p)—C(O)—R^(4a), —S(O)₂R^(4a), —NO₂, ortetrazolyl. In one specific embodiment, n is 1, 2, or 3 and each R¹ isthe same or different and independently H, halo, methyl, or ethyl. Incertain specific embodiments, R¹ is H at each occurrence. In otherspecific embodiments, n is 1 and R¹ is unsubstituted C₁-C₆ alkyl orhalo. In other specific embodiments, R¹ is unsubstituted C₁-C₆ alkyl andn is 1. In a specific embodiment, n is 1 and R¹ is C₁-C₃ alkyl, which inmore specific embodiments is unsubstituted. In another embodiment, n is1 and R¹ is halo, which in certain embodiments is chloro, fluoro, iodo,or bromo. In another particular embodiment, n is 1 and R¹ is methyl orethyl. In still another particular embodiment, n is 1 and R¹ is methyl.In certain embodiments, at least one R¹ is methyl or ethyl and theremaining R¹ are each H. In still another specific embodiment n is atleast 1 and the at least one R¹ is haloalkyl. In still other specificembodiments, n is 1 or 2 and each R¹ is haloalkyl. In still anotherspecific embodiment, n is 1 and R¹ is C₁-C₆ haloalkyl. In more specificembodiments, n is 1 and R¹ is C₁-C₃ haloalkyl. In another specificembodiment, n is 1 and R¹ is C₁-C₃ fluoroalkyl. In a more specificembodiment, the fluoroalkyl is a trifluoromethyl (—CF₃) or —CH₂CH₂CF₃.In another specific embodiment, n is 1 and R¹ is trifluoromethyl (—CF₃).In still another specific embodiment, n is 1 and R¹ is —CH₂CF₂CF₃. Inother certain embodiments, n is 1 and R¹ is —NO₂. In still other certainembodiments, n is 1 and R¹ is tetrazolyl (e.g., 5-tetrazolyl). In yetother certain embodiments, n is 1 and R¹ is —(CH₂)_(p)—C(O)—R^(4a). Inyet other certain embodiments, n is 1 and R¹ is —S(O)₂R^(4a).

In still another specific embodiment, n is 1 or 2, and at least one ofR¹ is —S(O)₂R^(4a) or —(CH₂)_(p)—C(O)—R^(4a), and R^(4a) is —OR⁷,—NR⁷R⁸, or —O(CH₂)_(q)—OC(O)R⁷. In other certain embodiments, at leastone of R¹ is —S(O)₂R^(4a) or —(CH₂)_(p)—C(O)—R^(4a), and R^(4a) is anamino acid residue. In another certain embodiment, at least one of R¹ is—S(O)₂R^(4a) or —(CH₂)_(p)—C(O)—R^(4a), and R^(4a) is a peptide thatconsists of two (i.e., a dipeptide), three (i.e., a tripeptide), four,five, or six amino acid residues. In particular embodiments, R⁷ is H orC₁-C₆ alkyl. In yet another specific embodiment, R⁷ is C₁-C₃ alkyl,which in particular embodiments is unsubstituted, and which in otherparticular embodiments is methyl or ethyl. In still another specificembodiment, R⁷ is a saccharide. In particular embodiments, R⁸ is H orC₁-C₆ alkyl. In yet another specific embodiment, R⁸ is C₁-C₃ alkyl,which in particular embodiments is unsubstituted, and which in otherparticular embodiments is methyl or ethyl. In still another specificembodiment, R⁸ is a saccharide. In certain embodiments, R^(4a) is—NR⁷R⁸, and each of R⁷ and R⁸ is the same or different and independentlyH or C₁-C₆ alkyl. In certain embodiments, R^(4a) is —NR⁷R⁸, and each ofR⁷ and R⁸ is the same or different and independently H, methyl, orethyl. In other particular embodiments, R^(4a) is —OR⁷ or—O(CH₂)_(q)—OC(O)R⁷ and R⁷ is an amino acid residue. In still othercertain embodiments, R^(4a) is —OR⁷ or —O(CH₂)_(q)—OC(O)R⁷ and R⁷ is apeptide that consists of two (i.e., a dipeptide), three (i.e., atripeptide), four, five, or six amino acid residues. In still othercertain embodiments, R^(4a) is —NR⁷R⁸ and either R⁷ or R⁸ is an aminoacid residue. In still other certain embodiments, R^(4a) is —NR⁷R⁸ andeither R⁷ or R⁸ is a peptide that consists of two (i.e., a dipeptide),three (i.e., a tripeptide), four, five, or six amino acid residues.

In particular embodiments, when R¹ is —(CH₂)_(p)—C(O)—R^(4a), p is 0, 1,or 2. In yet other certain embodiments, n is 1 and R¹ is—(CH₂)_(p)—C(O)—R^(4a), and R^(4a) is —OR⁷. In certain particularembodiments, R^(4a) is —OR⁷, and R⁷ is hydrogen, C₁-C₂₀ alkyl, or asaccharide. In other certain embodiments, when R¹ is—(CH₂)_(p)—C(O)—R^(4a), and R^(4a) is —OR⁷, R⁷ is hydrogen, methyl,ethyl. In other certain embodiments, when R¹ is —(CH₂)_(p)—C(O)—R^(4a),and R^(4a) is —OR⁷, R⁷ is a saccharide. In still another embodiment, R⁷is an amino acid residue. In still another particular embodiment, R⁷ isa peptide (i.e., 2-6 amino acids).

In another particular embodiment of the compound of structure (II), m is1 or 2. In another particular embodiment of the compound of structure(I), m is 1 or 2 and each R^(1a) is the same or different andindependently H, halo, haloalkyl, C₁-C₆ alkyl, —(CH₂)_(p)—C(O)—R^(4a),—S(O)₂R^(4a), —NO₂, or tetrazolyl. In one particular embodiment, m is 2and p is 0 and at least one of R^(1a) is halo, —NO₂, C₁-C₃ alkyl,—C(O)—R^(4a). In certain embodiments, R^(1a) is H at every occurrence.In particular embodiments, m is 1 or 2 and at least one of R^(1a) ishalo, which in particular embodiments halo is I, Cl, or Br. In morespecific embodiments, m is 1 or 2 and at least one of R^(1a) is Cl. Inother specific embodiments, m is 1 or 2 and R^(1a) is unsubstitutedC₁-C₆ alkyl. In a specific embodiment, m is 1 or 2 and at least oneR^(1a) is C₁-C₃ alkyl, which in more specific embodiments isunsubstituted. In another particular embodiment, m is 1 and R^(1a) ismethyl or ethyl. In still another particular embodiment, m is 1 andR^(1a) is methyl. In still other certain embodiments, m is 1 or 2, andat least one R^(1a) is tetrazolyl (e.g., 5-tetrazolyl). In anotherspecific embodiment, m is 1 or 2 and at least one of R^(1a) is —NO₂. Inmore specific embodiments, m is 1 and R^(1a) is C₁-C₃ haloalkyl. Inanother specific embodiment, m is 1 and R^(1a) is C₁-C₃ fluoroalkyl. Ina more specific embodiment, the fluoroalkyl is a trifluoromethyl (—CF₃)or —CH₂CH₂CF₃. In another specific embodiment, m is 1 and R^(1a) istrifluoromethyl (—CF₃). In still another specific embodiment, m is 1 andR^(1a) is —CH₂CF₂CF₃.

In still another specific embodiment, m is 1 or 2, and at least one ofR^(1a) is —(CH₂)_(p)—C(O)—R^(4a), and R^(4a) is —OR⁷, —NR⁷R⁸, or—O(CH₂)_(q)—OC(O)R⁷. In particular embodiments, when R^(1a) is—(CH₂)_(p)—C(O)—R^(4a), p is 0, 1, or 2. In one embodiment when R^(1a)is —(CH₂)_(p)—C(O)—R^(4a), R^(4a) is —OR⁷, and R⁷ is hydrogen, C₁-C₂₀alkyl, a saccharide, an amino acid residue, or a peptide (i.e., 2-6amino acids). In other particular embodiments, R⁷ is H or C₁-C₆ alkyl.In yet another specific embodiment, when R^(1a) is—(CH₂)_(p)—C(O)—R^(4a), and R^(4a) is R⁷ is C₁-C₃ alkyl, which inparticular embodiments is unsubstituted, and which in other particularembodiments is methyl or ethyl. In still another specific embodiment,when R^(1a) is —(CH₂)_(p)—C(O)—R^(4a) and R^(4a) is −OR⁷, R⁷ is asaccharide. In still other particular embodiments, R⁷ is an amino acidresidue. In still another particular embodiment, R⁷ is a peptide. Instill more specific embodiments, when R^(1a) is —(CH₂)_(p)—C(O)—R^(4a),R^(4a) is —OR⁷, p is 0, and R⁷ is H, methyl, or ethyl.

In still one specific embodiment when R^(1a) is —(CH₂)_(p)—C(O)—R^(4a),R^(4a) is —NR⁷R⁸, wherein each of R⁷ and R⁸ is the same or different andindependently hydrogen, C₁-C₆ alkyl, or a saccharide. In certainembodiments, when R^(4a) is —NR⁷R⁸, at least one or both of R⁷ and R⁸ ishydrogen. In other certain embodiments, when R^(4a) is —NR⁷R⁸, at leastone of R⁷ and R⁸ is hydrogen and the other is C₁-C₆ alkyl. In othercertain embodiments, when R^(4a) is —NR⁷R⁸, at least one of R⁷ and R⁸ ishydrogen and the other is C₁-C₃ alkyl, which in particular embodimentsis unsubstituted, and which in other particular embodiments is methyl orethyl. In yet another embodiment when R^(4a) is —NR⁷R⁸, at least one ofR⁷ and R⁸ is hydrogen and the other is a saccharide. In still otherparticular embodiments, R⁷ is an amino acid residue. In still anotherparticular embodiment, R⁷ is a peptide (i.e., consisting of 2, 3, 4, 5,or six amino acids).

In still another specific embodiment, when R^(1a) is—(CH₂)_(p)—C(O)—R^(4a), R^(4a) is —O(CH₂)_(q)—OC(O)R⁷ and R⁷ ishydrogen, C₁-C₂₀ alkyl, or a saccharide. In particular embodiments, p is1, 2, or 3; in other particular embodiments, q is 1, 2, or 3. In stillother specific embodiments, R⁷ is H or C₁-C₆ alkyl. In more specificembodiments, R⁷ is C₁-C₃ alkyl, which in particular embodiments isunsubstituted, and which in other particular embodiments is methyl orethyl. In another embodiment, R⁷ is a saccharide. In still otherparticular embodiments, R⁷ is an amino acid residue. In still anotherparticular embodiment, R⁷ is a peptide (i.e., consisting of 2, 3, 4, 5,or six amino acids).

In a particular embodiment, at least one of R¹ or R^(1a) is a polargroup selected from —NO₂, tetrazolyl, —S(O)₂OR⁷, or —C(═O)OR⁷ (whereinR⁷ is hydrogen, C₁₋₂₀ alkyl, or a saccharide). In specific embodiments,R⁷ is H or C₁-C₆ alkyl. In more specific embodiments, R⁷ is C₁-C₃ alkyl,which in particular embodiments is unsubstituted, and which in otherparticular embodiments is methyl or ethyl. In another embodiment, R⁷ isa saccharide. In still other particular embodiments, R⁷ is an amino acidresidue. In still another particular embodiment, R⁷ is a peptide.

In other particular embodiments, R^(2a) or R^(2b) are each the same ordifferent and independently hydrogen or C₁-C₃ alkyl. In yet anotherparticular embodiments, R^(2a) or R^(2b) are each the same or differentand independently hydrogen, methyl, or ethyl. In certain embodiments,R^(2a) and R^(2b) are each H. In other certain embodiments, R^(2a) andR^(2b) are each methyl. In still other certain embodiments, R^(2a) andR^(2b) are each ethyl.

In still another embodiment of the compound of structure (II), R⁴ is H,—N(═O), or unsubstituted C₁₋₆ alkyl. In another specific embodiment, R⁴is H, —N(═O), or methyl. In another specific embodiment of the compoundof structure (II), R⁴ is H. In another specific embodiment, R⁴ is—N(═O). In another specific embodiment, R⁴ is C₁-C₃ alkyl. In yetanother specific embodiment, R⁴ is methyl (—CH₃) or ethyl (—CH₂CH₃). Instill another specific embodiment, R⁴ is methyl.

In another specific embodiment of the compound of structure (II), R⁵ isH. In another specific embodiment, R⁵ is C₁-C₃ alkyl, which inparticular embodiments is unsubstituted. In one embodiment, R⁵ is H ormethyl. In still another specific embodiment, R⁵ is methyl. In a morespecific embodiment, each of R⁵ and R⁶ is methyl.

In yet another specific embodiment of the compound of structure (II), R⁶is C₁-C₃ alkyl, C₁-C₃ fluoroalkyl, chloro, bromo, iodo, or fluoro. Inanother specific embodiment, R⁶ is methyl, ethyl, trifluoromethyl,chloro, bromo, or iodo. In another specific embodiment, R⁶ is C₁-C₆alkyl. In another more specific embodiment, R⁶ is C₁-C₃ alkyl. In morespecific embodiments, R⁶ is unsubstituted C₁-C₆ alkyl. In yet anotherspecific embodiment, R⁶ is unsubstituted C₁-C₃ alkyl (i.e., —CH₃,—CH₂CH₃, or —CH₂CH₂CH₃ (branched or straight chain)). In still morespecific embodiments, R⁶ is methyl or ethyl. In still another morespecific embodiment, R⁶ is methyl. In yet another specific embodiment,R⁶ is C₁-C₆ haloalkyl. In more specific embodiments, R⁶ is C₁-C₃haloalkyl. In another specific embodiment, R⁶ is C₁-C₃ fluoroalkyl. In amore specific embodiment, the fluoroalkyl is a trifluoromethyl (—CF₃) or—CH₂CH₂CF₃. In another specific embodiment, R⁶ is trifluoromethyl(—CF₃). In still another specific embodiment, R⁶ is —CH₂CF₂CF₃. In yetanother particular embodiment, R⁶ is halo. In a more particularembodiment, R⁶ is chloro, bromo, or iodo. In still another particularembodiment, R⁶ is chloro. In yet another particular embodiment, R⁶ isbromo. In yet another particular embodiment, R⁶ is iodo.

With respect to the embodiments of structure (II) described above, whenR⁴ is H, R⁶ is ethyl. In certain particular embodiments of the compoundof structure (II), when R⁴ is H, R⁶ is ethyl. In certain particularembodiments, when R⁴ is H, R⁶ is halo, which in particular embodimentsis Cl, Br, or I. In other particular embodiments, when R⁴ is H, R⁶ is atrihaloalkyl, which in certain embodiments, is a trifluoroalkyl. In morespecific embodiments, when R⁴ is H, R⁶ is trifluoromethyl (—CF₃).

In other certain embodiments of the compound of structure (II), when R⁴is H, and R⁶ is methyl, then X is S. In still other certain embodiments,when R⁴ is H, and R⁶ is methyl, then R⁵ is also methyl.

In specific embodiments when R⁶ is —CH₃, then (i) R⁴ is —N(═O) or —CH₃;(ii) R⁵ is halo or C₁-C₆ alkyl; or (iii) X is S. In another specificembodiment when X is O, and each of R⁴ and R⁵ is H, then R⁶ is C₂-C₆alkyl, halo, or C₁-C₆ haloalkyl.

In further particular embodiments, the compound of structure (II) is anisolated R form.

In further particular embodiments, the compound of structure (II) is anisolated S form.

In another embodiment, the compound of structure II has the followingsubstructure, wherein R^(2a) and R^(2b) are each methyl, p is 0, R^(4a)is —OR⁷, n is 1, and R¹ is meta to the linking carbon, and the compoundhas the following structure (IIA):

wherein:

X is O or S;

R¹ is H, halo, or C₁-C₃ alkyl;

R² is H, halo, —NO₂, or —C(═O)OR⁷;

R³ is H or NO₂;

R⁴ is —N(═O), C₁-C₃ alkyl, or H;

R⁵ is H or C₁-C₃ alkyl;

R⁶ is C₁-C₆ alkyl, C₁-C₆ haloalkyl, or halo; and

R⁷ is H, C₁-C₆ alkyl, a saccharide, or an amino acid residue, or apeptide.

The following compounds as shown in Table A are excluded from thecompounds that have the structure (IIA):

(a)7,9-Dimethyl-11-(3-methylphenyl)-6-(5-methylfuran-2-yl)-5,6-dihydro-pyrimido[4′,5′-3,4]pyrrolo[1,2-a]quinoxaline-8,10-(7H,9H)-dione;

(b)7,9-Dimethyl-11-phenyl-6-(5-methylfuran-2-yl)-5,6-dihydro-pyrimido[4′,5′-3,4]pyrrolo[1,2-a]quinoxaline-8,10-(7H,9H)-dione;and

(d)2,3,7,9-Tetramethyl-11-phenyl-6-(5-methylfuran-2-yl)-5,6-dihydro-pyrimido[4′,5′-3,4]pyrrolo[1,2-a]quinoxaline-8,10-(7H,9H)-dione.

In certain embodiments of the compound of structure (IIA), X is O. Inother certain embodiments of the compound of structure (IIA), X is S.

In more specific embodiments of the compound of structure (IIA), R¹ isH, —CH₃, or —CH₂CH₃. In a more particular embodiment, R¹ is H or —CH₃.In other particular embodiments, R¹ is H. In still another embodiment,R¹ is —CH₃. In yet another embodiment, R¹ is —CH₂CH₃. In still anotherspecific embodiment, R¹ is halo, which in particular embodiments is anyone of Cl, F, I, or Br.

In more specific embodiments of the compound of structure (IIA), R² isH, chloro, —NO₂, or —C(═O)OR⁷, wherein R⁷ is H or C₁-C₆ alkyl. In stillanother specific embodiment, R² is H, chloro, —NO₂, or —C(═O)OR⁷,wherein R⁷ is H, —CH₃, —CH₂CH₃, or —CH₂CH₂CH₃. In one particularembodiment, R² is H, chloro, or —NO₂. In another particular embodiment,R² is or —C(═O)OR⁷, wherein R⁷ is H, —CH₃, —CH₂CH₃, or —CH₂CH₂CH₃. In aparticular embodiment, R² is H. In another embodiment, R² is H, chloro,—NO₂, or —C(═O)OR⁷, wherein R⁷ is H, —CH₃, or —CH₂CH₃. In anotherspecific embodiment, R² is halo, which in particular embodiments is I,Cl, or Br. In more specific embodiments, R² is Cl. In another specificembodiment, R² is —NO₂. In still another specific embodiment, R² is—C(═O)OR⁷ and R⁷ is methyl. In a more particular embodiment, R² is—C(═O)OR⁷, and R⁷ is H. In still another specific embodiment, R² is—C(═O)OR⁷, and R⁷ is ethyl. In still other particular embodiments, R⁷ isan amino acid residue. In still another particular embodiment, R⁷ is apeptide (i.e., consisting of 2, 3, 4, 5, or six amino acids). In yetanother embodiment, R⁷ is a saccharide.

In another particular embodiment of the compound of structure (IIA), R³is H. In another specific embodiment, R³ is —NO₂.

In another specific embodiment of the compound of structure (IIA), R⁴ isH, methyl, or —N(═O). In another specific embodiment R⁴ is H. In anotherspecific embodiment, R⁴ is —N(═O). In one specific embodiment, R⁴ isethyl. In yet another specific embodiment, R⁴ is —CH₃.

In another specific embodiment of the compound of structure (IIA), R⁵ isH. In another specific embodiment, R⁵ is C₁-C₃ alkyl, which inparticular embodiments is unsubstituted. In one embodiment, R⁵ is H ormethyl. In still another specific embodiment, R⁵ is methyl (—CH₃). In amore specific embodiment, each of R⁵ and R⁶ is methyl.

In yet another specific embodiment of the compound of structure (IIA),R⁶ is C₁-C₃ alkyl, C₁-C₃ fluoroalkyl, chloro, bromo, iodo, or fluoro. Inanother specific embodiment, R⁶ is methyl, ethyl, trifluoromethyl,chloro, bromo, or iodo. In another specific embodiment, R⁶ is C₁-C₆alkyl. In another more specific embodiment, R⁶ is C₁-C₃ alkyl. In morespecific embodiments, R⁶ is unsubstituted C₁-C₆ alkyl. In yet anotherspecific embodiment, R⁶ is unsubstituted C₁-C₃ alkyl (i.e., —CH₃,—CH₂CH₃, or —CH₂CH₂CH₃ (branched or straight chain)). In still morespecific embodiments, R⁶ is methyl or ethyl. In still another morespecific embodiment, R⁶ is methyl. In yet another specific embodiment,R⁶ is C₁-C₆ haloalkyl. In more specific embodiments, R⁶ is C₁-C₃haloalkyl. In another specific embodiment, R⁶ is C₁-C₃ fluoroalkyl. In amore specific embodiment, the fluoroalkyl is a trifluoromethyl (—CF₃) or—CH₂CH₂CF₃. In another specific embodiment, R⁶ is trifluoromethyl(—CF₃). In still another specific embodiment, R⁶ is —CH₂CF₂CF₃. In yetanother particular embodiment, R⁶ is halo. In a more particularembodiment, R⁶ is chloro, bromo, or iodo. In still another particularembodiment, R⁶ is chloro. In yet another particular embodiment, R⁶ isbromo. In yet another particular embodiment, R⁶ is iodo.

In further particular embodiments, the compound of structure (IIA) is anisolated R form.

In further particular embodiments, the compound of structure (IIA) is anisolated S form.

In certain specific embodiments, PPQ compounds of structure (II),substructure (IIA), are as follows:

Compound Designation Compound Structure Compound Name PPQ-4

7,9-Dimethyl-6-(5- methylfuran-2-yl)-5- nitroso-11-phenyl-5,6-dihydropyrimido[4′,5′- 3,4] pyrrolo[1,2- a]quinoxaline-8,10-(7H,9H)-dione PPQ-5

5,7,9-Trimethyl-6-(5- methylfuran-2-yl)-11- phenyl-5,6- dihydropyrimido[4′,5′- 3,4] pyrrolo[1,2- a]quinoxaline-8,10- (7H,9H)-dione PPQ-6

7,9-Dimethyl-6-(5- ethylfuran-2-yl)-11- phenyl-5,6-dihydropyrimido[4′,5′- 3,4] pyrrolo[1,2- a]quinoxaline-8,10-(7H,9H)-dione PPQ-7

7,9-Dimethyl-6-(5- methylthiophene-2-yl)- 11-phenyl-5,6-dihydropyrimido[4′,5′- 3,4] pyrrolo[1,2- a]quinoxaline-8,10-(7H,9H)-dione PPQ-8

7,9-Dimethyl-6-(4,5- dimethylfuran-2-yl)-11- phenyl-5,6-dihydropyrimido[4′,5′- 3,4] pyrrolo[1,2- a]quinoxaline-8,10-(7H,9H)-dione PPQ-9

6-(5-Chlorofuran-2-yl)- 7,9-dimethyl-11- phenyl-5,6-dihydropyrimido[4′,5′- 3,4] pyrrolo[1,2- a]quinoxaline-8,10-(7H,9H)-dione PPQ-10

6-(5-Bromofuran-2-yl)- 7,9-dimethyl-11- phenyl-5,6-dihydropyrimido[4′,5′- 3,4] pyrrolo[1,2- a]quinoxaline-8,10-(7H,9H)-dione PPQ-11

7,9-Dimethyl-6-(5- iodofuran-2-yl)-11- phenyl-5,6-dihydropyrimido[4′,5′- 3,4]pyrrolo[1,2- a]quinoxaline-8,10-(7H,9H)-dione PPQ-12

7,9-Dimethyl-11- phenyl-6-(5- trifluoromethylfuran-2- yl)-5,6-dihydropyrimido[4′,5′- 3,4] pyrrolo[1,2- a]quinoxaline-8,10-(7H,9H)-dione PPQ-23

6-(5-Bromofuran-2-yl)- 7,9-dimethyl-2-nitro- 11-phenyl-5,6-dihydropyrimido[4′,5′- 3,4] pyrrolo[1,2- a]quinoxaline-8,10-(7H,9H)-dione

As discussed in greater detail herein, also provided are pharmaceuticalcompositions comprising any one or more of the above-described BPO(i.e., the compounds of structure I and substructures (Ia(i)), (Ib(i)),(IA), (IA1), (IB), (IB 1), (IC), (IC1), and specific compounds) and PPQcompounds (i.e., the compounds of structure (II), substructure IIA, andspecific compounds) and a pharmaceutically (i.e., physiologically)suitable (i.e., acceptable) excipient (such as a diluent, carrier, oradjuvant). The BPO and PPQ compounds having the structures describedherein are capable of inhibiting (i.e., slowing, retarding, decreasing,reducing) CFTR-mediated ion transport (i.e., inhibiting in astatistically significant, clinically significant, and/or biologicallysignificant manner), for example, inhibiting CFTR-mediated chloride ion(i.e., Cl⁻) transport. In other embodiments provided herein, the BPO andPPQ compounds and compositions comprising these compounds describedabove and herein may be used in methods for treating a disease,condition, or disorder that is treatable by inhibiting CFTR-mediated iontransport. Exemplary diseases, conditions, and disorders include, butare not limited to, polycystic kidney disease (PKD or PCKD) (includingautosomal dominant PKD and autosomal recessive PKD), aberrantlyincreased intestinal fluid secretion, and secretory diarrhea. Inparticular embodiments, the BPO and PPQ compounds and compositionscomprising any one or more of the BPO and PPQ compounds may be used inmethods for inhibiting (i.e., preventing, delaying, slowing, retarding)cyst formation (i.e., reducing the likelihood of occurrence of one ormore cysts forming) and/or inhibiting cyst enlargement or expansion(i.e., slowing, reducing, preventing, retarding, reversing, decreasingcyst enlargement or expansion), particularly inhibiting cyst formationor inhibiting cyst enlargement in one or both kidneys of a human ornon-human animal. Inhibiting cyst enlargement or expansion may thusreduce or decrease the volume of one or more fluid-filled cysts. Each ofthese methods and uses is described in greater detail herein.

Definitions

Certain chemical groups named herein are preceded by a shorthandnotation indicating the total number of carbon atoms that are to befound in the indicated chemical group. For example, C₁₋₆ alkyl (or C₁-C₆alkyl) describes an alkyl group, as defined below, has a total of 1, 2,3, 4, 5, or 6 carbon atoms. Similarly, C₁₋₃ alkyl (or C₁-C₃ alkyl)describes an alkyl group, as defined below, has a total of 1, 2, or 3carbon atoms. By way of additional example, C₁₋₂₀ alkyl (or C₁-C₂₀alkyl) describes an alkyl group, as defined below, has a total of anynumber of carbon atoms between 1 and 20 (i.e., 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14 15, 16, 17, 18, 19, or 20 carbon atoms). The totalnumber of carbons in the shorthand notation does not include carbonsthat may exist in substituents of the group described. In addition tothe foregoing, as used herein, unless specified to the contrary, thefollowing terms have the meaning indicated.

“Alkyl” means a straight chain or branched, noncyclic or cyclic,saturated aliphatic hydrocarbon containing from 1 to 20 carbon atoms,while the terms “C₁₋₆ alkyl” and “C₁₋₃ alkyl” have the same meaning asalkyl but contain from 1 to 6 carbon atoms or 1 to 3 carbon atoms,respectively. Representative saturated straight chain alkyls includemethyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, and the like, whilesaturated branched alkyls include isopropyl, sec-butyl, isobutyl,tert-butyl, isopentyl, and the like. Representative saturated cyclicalkyls (e.g., C₃₋₂₀ cycloalkyl) include cyclopropyl, cyclobutyl,cyclopentyl, cyclohexyl, —CH₂cyclopropyl, —CH₂cyclobutyl,—CH₂cyclopentyl, —CH₂cyclohexyl, and the like. Cyclic alkyls, alsoreferred to as “homocyclic rings,” include di- and poly-homocyclic ringssuch as decalin and adamantyl. A straight or branched hydrocarbon chainradical group may contain at least one double or triple bond betweenadjacent carbon atoms (referred to as an “alkenyl” or “alkynyl,”respectively). Representative straight chain and branched alkenyls(e.g., C₂₋₆ alkenyl) include ethylenyl, propylenyl, 1-butenyl,2-butenyl, isobutylenyl, 1-pentenyl, 2-pentenyl, 3-methyl-1-butenyl,2-methyl-2-butenyl, 2,3-dimethyl-2-butenyl, and the like; representativestraight chain and branched alkynyls include acetylenyl, propynyl,1-butynyl, 2-butynyl, 1-pentynyl, 2-pentynyl, 3-methyl-1 butynyl, andthe like. Unless otherwise specified, it is understood that within thecontext of the current disclosure, the term “alkyl” can be optionallysubstituted, i.e., “optionally substituted alkyl” encompassesunsubstituted alkyl and substituted alkyl as defined herein.

As used herein, the term “substituted” in the context of alkyl, alkenyl,aryl, heteroaryl, and alkoxy means that at least one hydrogen atom ofthe alky, aryl, and heteroaryl moiety is replaced with a substituent. Inthe instance of an oxo substituent (“═O”) two hydrogen atoms arereplaced. A “substituent” as used within the context of this disclosureincludes oxo, halogen, hydroxy, cyano, nitro, amino, alkylamino,dialkylamino, alkyl, alkoxy, thioalkyl, haloalkyl, substituted alkyl,heteroalkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl,heteroaryl, substituted heteroaryl, heteroarylalkyl, substitutedheteroarylalkyl, heterocycle, substituted heterocycle, heterocyclealkyl,substituted heterocyclealkyl, —NR_(a)R_(b), —NR_(a)C(═O)R_(b),—NR_(a)C(═O)NR_(a)R_(b), —NR_(a)C(═O)OR_(b)—NR_(a)S(═O)₂R_(b), —OR_(a),—C(═O)R_(a)—C(═O)OR_(a), —C(═O)NR_(a)R_(b), —OCH₂C(═O)NR_(a)R_(b),—OC(═O)NR_(a)R_(b), —SH, —SR_(a), —SOR_(a), —S(═O)₂NR_(a)R_(b),—S(═O)₂R_(a), —SR_(a)C(═O)NR_(a)R_(b), —OS(═O)₂R_(a) and —S(═O)₂OR_(a),wherein R_(a) and R_(b) are the same or different and independentlyhydrogen, alkyl, haloalkyl, substituted alkyl, alkoxy, aryl, substitutedaryl, arylalkyl, substituted arylalkyl, heteroaryl, substitutedheteroaryl, heteroarylalkyl, substituted heteroarylalkyl, heterocycle,substituted heterocycle, heterocyclealkyl or substitutedheterocyclealkyl. As used herein and according to chemistry convention,the formula —C(═O)— may also be represented as —C(O)—, or —S(═O)₂— maybe represented as —S(O)₂—. By way of example, —C(═O)R_(a) has the samemeaning as —C(O)R_(a).

Representative substituents include (but are not limited to) alkoxy(i.e., alkyl-O—, e.g., methoxy, ethoxy, propoxy, butoxy, pentoxy),aryloxy (e.g., phenoxy, chlorophenoxy, tolyloxy, methoxyphenoxy,benzyloxy, alkyloxycarbonylphenoxy, alkyloxycarbonyloxy,acyloxyphenoxy), acyloxy (e.g., propionyloxy, benzoyloxy, acetoxy),carbamoyloxy, carboxy, mercapto, alkylthio, acylthio, arylthio (e.g.,phenylthio, chlorophenylthio, alkylphenylthio, alkoxyphenylthio,benzylthio, alkyloxycarbonyl-phenylthio), amino (e.g., amino, mono- anddi- C₁₋₃ alkanylamino, methylphenylamino, methylbenzylamino, C₁₋₃alkanylamido, acylamino, carbamamido, ureido, guanidino, nitro andcyano). Moreover, any substituent may have from 1-5 further substituentsattached thereto.

“Alkenyl” refers to a straight or branched, noncyclic or cyclic,hydrocarbon radical group consisting solely of carbon and hydrogenatoms, containing at least one double bond, and having from two totwelve carbon atoms. In certain embodiments, an alkenyl may comprise twoto eight carbon atoms. In other embodiments, an alkenyl may comprise twoto six carbon atoms (i.e., C₂-C₆ alkenyl). In other embodiments, analkenyl may comprise two to four carbon atoms. The alkenyl is connectedto the rest of the molecule by a single bond, for example, ethenyl(i.e., vinyl), prop-1-enyl (i.e., allyl), but-1-enyl, pent-1-enyl,penta-1,4-dienyl, and the like. Representative straight chain andbranched alkenyls (e.g., C₂₋₆ alkenyl) include ethylenyl, propylenyl,1-butenyl, 2-butenyl, isobutylenyl, 1-pentenyl, 2-pentenyl,3-methyl-1-butenyl, 2-methyl-2-butenyl, 2,3-dimethyl-2-butenyl, and thelike. Representative unsaturated cyclic alkyls include cyclopentenyl andcyclohexenyl, and the like. Unless otherwise specified, it is understoodthat within the context of the current disclosure, the term “alkenyl”can be optionally substituted, i.e., “optionally substituted alkenyl”encompasses unsubstituted alkyl and substituted alkenyl as definedherein.

“Aryl” refers to aromatic monocyclic or multicyclic hydrocarbon ringsystem consisting only of hydrogen and carbon and containing from six toeighteen carbon atoms, where the ring system may be partially or fullysaturated. Aryl groups include, but are not limited to, groups such asfluorenyl, phenyl and naphthyl. Unless otherwise specified, it isunderstood that within the context of the current disclosure, the term“aryl” can be optionally substituted, i.e., “optionally substitutedaryl” encompasses unsubstituted aryl and substituted aryl (e.g.,substituted phenyl) as defined herein.

“Haloalkyl” refers to an alkyl radical, as defined above, that issubstituted by one or more halo radicals, as defined above, for example,trifluoromethyl, difluoromethyl, trichloromethyl, 2,2,2-trifluoroethyl,1-fluoromethyl-2-fluoroethyl, 3-bromo-2-fluoropropyl,1-bromomethyl-2-bromoethyl, and the like. The alkyl part of thehaloalkyl radical may be optionally substituted as defined above for analkyl group.

“Heteroaryl” means an aromatic heterocycle ring of 5- to 10 members andhaving at least one heteroatom selected from nitrogen, oxygen andsulfur, and containing at least 1 carbon atom, including monocyclic,bicyclic and tricyclic ring systems. A fused heteroaryl (e.g., abicyclic heteroaryl) contains at least one aromatic ring, which may be abenzo ring (e.g., benzofuranyl, 1,3-benzodioxolyl or indolyl).Representative heteroaryls are furanyl, benzofuranyl, thienyl,benzothienyl, 1,3-benzodioxolyl, pyrrolyl, indolyl, isoindolyl,azaindolyl, pyridyl, quinolinyl, isoquinolinyl, oxazolyl, isooxazolyl,benzoxazolyl, pyrazolyl, imidazolyl, benzimidazolyl, thiazolyl,benzothiazolyl, isothiazolyl, pyridazinyl, pyrimidinyl, pyrazinyl,triazinyl, cinnolinyl, phthalazinyl, and quinazolinyl. Unless otherwisespecified, it is understood that within the context of the currentdisclosure, the term “heteroaryl” can be optionally substituted, i.e.,“optionally substituted heteroaryl” encompasses unsubstituted heteroaryland substituted heteroaryl (e.g., substituted furanyl) as definedherein.

“Heterocycle” (also referred to herein as a “heterocyclic ring”) means a4- to 7-membered monocyclic, or 7- to 10-membered bicyclic, heterocyclicring which is either saturated, unsaturated, or aromatic, and whichcontains from 1 to 4 heteroatoms independently selected from nitrogen,oxygen and sulfur, and wherein the nitrogen and sulfur heteroatoms maybe optionally oxidized, and the nitrogen heteroatom may be optionallyquaternized, including bicyclic rings in which any of the aboveheterocycles are fused to a benzene ring. The heterocycle may beattached via any heteroatom or carbon atom. Heterocycles includeheteroaryls as defined above. Thus, in addition to the heteroarylslisted above, heterocycles also include morpholinyl, pyrrolidinonyl,pyrrolidinyl, piperidinyl, hydantoinyl, valerolactamyl, oxiranyl,oxetanyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahydropyridinyl,tetrahydroprimidinyl, tetrahydrothiophenyl, tetrahydrothiopyranyl,tetrahydropyrimidinyl, tetrahydrothiophenyl, tetrahydrothiopyranyl, andthe like.

“Halogen” or “halo” means fluoro (F), chloro (Cl), bromo (B), and iodo(I).

“Alkoxy” refers to the radical: —O-alkyl, such as methoxy, ethoxy, andthe like. C₁₋₆alkoxy means that the alkyl moiety is C₁₋₆ alkyl.

A saccharide includes any monosaccharide, disaccharide, trisaccharide,or polysaccharide. In particular embodiments, such a saccharidecontributes to the polarity of the compound. Exemplary saccharidesinclude those to which a host is commonly exposed and thereby hasreduced immunogenicity in the host subject.

“Amino acid residue” refers to the portion of an amino acid that remainsafter losing a water molecule (or alcohol) when the amino acid iscondensed with a molecule. Typically, an amino acid is condensed with amolecule, including a compound of any of structure (I) or (II) (orsubstructures or specific structures), by forming a peptide bond. Incertain embodiments, the amino functional group of the amino acid can becondensed with a carboxylic acid group or its reactive equivalent (e.g.,carboxylic anhydride) of the molecule. In other embodiments, thecarboxylic acid functional group of the amino acid can be condensed withan amine group of the molecule. Typically, a molecule of water is lostduring the formation of the peptide bond. Examples of the “amino acidresidues” include, but are not limited to, residues of alanine,arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid,glycine, histidine, isoleucine, leucine, lysine, methionine,phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine,phosphoserine, phosphothreonine, phosphotyrosine, 4-hydroxyproline,hydroxylysine, demosine, isodemosine, gamma-carboxyglutamate, hippuricacid, octahydroindole-2-carboxylic acid, statine,1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, penicillamine,ornithine, 3-methylhistidine, norvaline, beta-alanine,gamma-aminobutylic acid, cirtulline, homocysteine, homoserine,methyl-alanine, para-benzoylphenylalanine, phenylglycine,propargylglycine, sarcosine, methionine sulfone, tert-butylglycine,3,5-dibromotyrosine, 3,5-diiodotyrosine, glycosylated threonine,glyclosylated serine, and glycosylated asparagine. A “peptide” as usedherein consists of any number of amino acids between 2 and 6 (i.e., 2,3, 4, 5, or 6 amino acids) linked together by peptide bonds. At leastone terminal end of the peptide has an amino acid residue as definedabove that is capable of being covalently linked to the compound. Ascommonly used in the art, the term “dipeptide” refers to a peptide thathas two amino acids, and “tripeptide” refers to a peptide that has threeamino acids. A single amino acid residue or peptide may be bonded to anycompound described herein to facilitate (i.e., promote) or to improvethe capability of the compound to enter a cell (e.g., an enterocyte). Inother words the amino acid residue or peptide may bind to a molecule(e.g., a cell receptor) that is present on the outer cell membrane andwhich may facilitate (i.e., promote) uptake or enhance uptake of thecompound.

The compounds described herein may generally be used as the free acid orfree base. Alternatively, the compounds may be used in the form of acidor base addition salts. Acid addition salts of the free base aminocompounds may be prepared according to methods well known in the art,and may be formed from organic and inorganic acids. Suitable organicacids include (but are not limited to) maleic, fumaric, benzoic,ascorbic, succinic, methanesulfonic, acetic, oxalic, propionic,tartaric, salicylic, citric, gluconic, lactic, mandelic, cinnamic,aspartic, stearic, palmitic, glycolic, glutamic, and benzenesulfonicacids. Suitable inorganic acids include (but are not limited to)hydrochloric, hydrobromic, sulfuric, phosphoric, and nitric acids. Baseaddition salts of the free acid compounds of the compounds describedherein may also be prepared by methods well known in the art, and may beformed from organic and inorganic bases. Suitable inorganic basesincluded (but are not limited to) the hydroxide or other salt of sodium,potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper,manganese, aluminum, and the like, and organic bases such as substitutedammonium salts. Thus, the term “pharmaceutically acceptable salt” ofcompounds of Structures I and II and substructures thereof, as well asany and all substructures and specific compounds described herein isintended to encompass any and all pharmaceutically suitable salt forms.

Compounds of Structures I and II and substructures thereof may sometimesbe depicted as an anionic species. One of ordinary skill in the art willrecognize that the compounds exist with an equimolar ratio of cation.For instance, the compounds described herein can exist in the fullyprotonated form, or in the form of a salt such as sodium, potassium,ammonium or in combination with any inorganic base as described above.When more than one anionic species is depicted, each anionic species mayindependently exist as either the protonated species or as the saltspecies. In some specific embodiments, the compounds described hereinexist as the sodium salt.

“Prodrug” is meant to indicate a compound that may be converted underphysiological conditions or by solvolysis to a biologically activecompound as described herein. Thus, the term “prodrug” refers to ametabolic precursor of a BPO or PPQ compound that is pharmaceuticallyacceptable. A prodrug may be inactive when administered to a subject inneed thereof, but is converted in vivo to an active compound. Prodrugsare typically rapidly transformed in vivo to yield the parent compound,for example, by hydrolysis in blood. The prodrug compound often offersadvantages of solubility, druggability, tissue compatibility or delayedrelease in a mammalian organism (see, e.g., Bundgard, H., Design ofProdrugs (1985), pp. 7-9, 21-24 (Elsevier, Amsterdam).

A discussion of prodrugs is also provided in Higuchi, T., et al.,“Pro-drugs as Novel Delivery Systems,” A.C.S. Symposium Series, Vol. 14,and in Bioreversible Carriers in Drug Design, ed. Edward B. Roche,American Pharmaceutical Association and Pergamon Press, 1987, both ofwhich are incorporated in full by reference herein.

The term “prodrug” is also meant to include any covalently bondedcarriers, which release the active compound in vivo when such prodrug isadministered to a subject. Prodrugs of a compound described herein maybe prepared by modifying functional groups present in the compound insuch a way that the modifications are cleaved, either in routinemanipulation or in vivo, to the parent compound. Prodrugs includecompounds described herein, wherein a hydroxy, amino, or mercapto groupis bonded to any group that, when the prodrug of the compound isadministered to a subject, cleaves to form a free hydroxy, free amino,or free mercapto group, respectively. Examples of prodrugs include, butare not limited to, acetate, formate, and benzoate derivatives ofalcohol or amine functional groups in the compounds and the like.

With regard to stereoisomers, the compounds of structure (I) andstructure (II), as well as any sub-structure herein (e.g., IA, IB, IC,IIA and other substructures), may have one or more chiral (orasymmetric) centers, for example, at the 6 position of thepyrimido[4′,5′-3,4]pyrrolo[1,2-a]quinoxaline ring system, and may thusgive rise to enantiomers, diastereomers, and other stereoisomeric formsthat may be defined, in terms of absolute stereochemistry, as (R)— or(S)—, at a given chiral center.

Using structure (I) as an example, unless otherwise specified, thecompounds of structure (I) (and Structure (II)) are not limited to anyabsolute stereochemistry. Thus, all possible isomers, includingdiastereomers and racemates, as well as optically pure forms (asisolated R or S form), and all tautomeric forms are intended to beincluded.

It is further specifically contemplated that “enantiomers,” which refersto two stereoisomers whose molecules are nonsuperimposeable mirrorimages of one another, may be isolated in either R or S form (shownbelow). As used herein, an isolated enantiomer (i.e., an isolated R or Sform) has an enantiomeric excess, which is defined as the absolutedifference between the mole fraction of the isolated enantiomer and itsopposite enantiomer, wherein the mole fraction of the isolatedenantiomer is of at least 85%, at least 90%, at least 95%, at least 98%,or at least 99%, or 100%.

Structure (I) as Isolated Enantiomers

When the compounds described herein contain olefinic double bonds orother centers of geometric asymmetry, and unless specified otherwise, itis intended that the compounds may be an isolated E or an isolated Z ora mixture of both E and Z geometric isomers (e.g., cis or trans).

Furthermore, some of the crystalline forms of any compound describedherein may exist as polymorphs, which are also included and contemplatedby the present disclosure. In addition, some of the compounds may formsolvates with water or other organic solvents. Such solvates aresimilarly included within the scope of compounds and compositionsdescribed herein.

In general, the compounds used in the reactions described herein may bemade according to organic synthesis techniques known to those skilled inthis art, starting from commercially available chemicals and/or fromcompounds described in the chemical literature. “Commercially availablechemicals” may be obtained from standard commercial sources includingAcros Organics (Pittsburgh Pa.), Aldrich Chemical (Milwaukee Wis.,including Sigma Chemical and Fluka), Apin Chemicals Ltd. (Milton ParkUK), Avocado Research (Lancashire U.K.), BDH Inc. (Toronto, Canada),Bionet (Cornwall, U.K.), Chemservice Inc. (West Chester Pa.), CrescentChemical Co. (Hauppauge N.Y.), Eastman Organic Chemicals, Eastman KodakCompany (Rochester N.Y.), Fisher Scientific Co. (Pittsburgh Pa.), FisonsChemicals (Leicestershire UK), Frontier Scientific (Logan Utah), ICNBiomedicals, Inc. (Costa Mesa Calif.), Key Organics (Cornwall U.K.),Lancaster Synthesis (Windham N.H.), Maybridge Chemical Co. Ltd.(Cornwall U.K.), Parish Chemical Co. (Orem Utah), Pfaltz & Bauer, Inc.(Waterbury Conn.), Polyorganix (Houston Tex.), Pierce Chemical Co.(Rockford Ill.), Riedel de Haen AG (Hanover, Germany), Spectrum QualityProduct, Inc. (New Brunswick, N.J.), TCI America (Portland Oreg.), TransWorld Chemicals, Inc. (Rockville Md.), and Wako Chemicals USA, Inc.(Richmond Va.).

Methods known to one of ordinary skill in the art may be identifiedthrough various reference books and databases. Suitable reference booksand treatise that detail the synthesis of reactants useful in thepreparation of compounds of the present disclosure, or providereferences to articles that describe the preparation, include forexample, “Synthetic Organic Chemistry,” John Wiley & Sons, Inc., NewYork; S. R. Sandler et al., “Organic Functional Group Preparations,” 2ndEd., Academic Press, New York, 1983; H. O. House, “Modern SyntheticReactions”, 2nd Ed., W. A. Benjamin, Inc. Menlo Park, Calif. 1972; T. L.Gilchrist, “Heterocyclic Chemistry”, 2nd Ed., John Wiley & Sons, NewYork, 1992; J. March, “Advanced Organic Chemistry: Reactions, Mechanismsand Structure,” 4th Ed., Wiley-Interscience, New York, 1992. Additionalsuitable reference books and treatise that detail the synthesis ofreactants useful in the preparation of compounds of the presentdisclosure, or provide references to articles that describe thepreparation, include for example, Fuhrhop, J. and Penzlin G. “OrganicSynthesis: Concepts, Methods, Starting Materials”, Second, Revised andEnlarged Edition (1994) John Wiley & Sons ISBN: 3-527-29074-5; Hoffman,R. V. “Organic Chemistry, An Intermediate Text” (1996) Oxford UniversityPress, ISBN 0-19-509618-5; Larock, R. C. “Comprehensive OrganicTransformations: A Guide to Functional Group Preparations” 2nd Edition(1999) Wiley-VCH, ISBN: 0-471-19031-4; March, J. “Advanced OrganicChemistry: Reactions, Mechanisms, and Structure” 4th Edition (1992) JohnWiley & Sons, ISBN: 0-471-60180-2; Otera, J. (editor) “Modern CarbonylChemistry” (2000) Wiley-VCH, ISBN: 3-527-29871-1; Patai, S. “Patai's1992 Guide to the Chemistry of Functional Groups” (1992) InterscienceISBN: 0-471-93022-9; Quin, L. D. et al. “A Guide to OrganophosphorusChemistry” (2000) Wiley-Interscience, ISBN: 0-471-31824-8; Solomon, T.W. G. “Organic Chemistry” 7th Edition (2000) John Wiley & Sons, ISBN:0-471-19095-0; Stowell, J. C., “Intermediate Organic Chemistry” 2ndEdition (1993) Wiley-Interscience, ISBN: 0-471-57456-2; “IndustrialOrganic Chemicals: Starting Materials and Intermediates: An Ullmann'sEncyclopedia” (1999) John Wiley & Sons, ISBN: 3-527-29645-X, in 8volumes; “Organic Reactions” (1942-2000) John Wiley & Sons, in over 55volumes; and “Chemistry of Functional Groups” John Wiley & Sons, in 73volumes.

Specific and analogous reactants may also be identified through theindices of known chemicals prepared by the Chemical Abstract Service ofthe American Chemical Society, which are available in most public anduniversity libraries, as well as through on-line databases (the AmericanChemical Society, Washington, D.C., may be contacted for more details).Chemicals that are known but not commercially available in catalogs maybe prepared by custom chemical synthesis houses, where many of thestandard chemical supply houses (e.g., those listed above) providecustom synthesis services. A reference for the preparation and selectionof pharmaceutical salts of the present disclosure is P. H. Stahl & C. G.Wermuth “Handbook of Pharmaceutical Salts,” Verlag Helvetica ChimicaActa, Zurich, 2002.

Preparation of the BPO Compounds

The following General Reaction Schemes illustrate methods to makecompounds of this disclosure, i.e., compounds of structures (I),(Ia(i)), (Ib(i)), (IA), (IB), (IC),

wherein R¹, R^(1a), R^(2a), R^(2b), R^(4a), R⁷, Z are described above,as an isolated enantiomer or a racemic mixture thereof, or apharmaceutically acceptable salt thereof. It is understood that in thefollowing Reaction Schemes, combinations of substituents and/orvariables of the depicted structures are permissible only if suchcontributions result in stable compounds.

Generally speaking, commercially available 6-methyluracil (1) can befirst alkylated to provide 1,3-dialkyl6-methyluracil (2). Suitablealkylating agents include, for example, alkylsulfate, etc. In particularembodiments, the alkylation is methylation and the exemplary alkylatingagents include dimethylsulfate, iodomethane, etc. Thereafter,1,3-dialkyl-6-methyluracil (2) is further acylated to provide ketone 4via, e.g., a Friedel-Crafts mechanism, in the presence of anappropriately substituted benzoyl chloride (3) and a Lewis acid catalyst(e.g., ZnCl). Ketone (4) can then be brominated to provide compound (5).Reaction of compound (5) and an appropriately substituted 2-aminophenol(5b) provides a ring-condensed product (5a). Compound (5a) can becombined with a formyl-substituted heteroaryl (furanyl or thienyl group)or a formyl-substituted aryl (6a) to produce a ring-condensed productrepresented by Structure (I).

In certain embodiments, the ring-condensed product is a racemic mixtureof enantiomers. Separation of the enantiomers may be carried out bychiral Supercritical Fluid Chromatography (SFC). The isolated enantiomermay be amorphous and can be further crystallized according to knownmethods in the art. The absolute enantiomer structure can be determinedby X-ray crystallography. For compounds having a carboxylic acidsubstituent (e.g., BPO-27), a suitable salt, amide or ester may beformed prior to crystallization in order to obtain crystals ofsufficient quality for X-ray crystallography.

Preparation of the PPQ Compounds

The following General Reaction Schemes illustrate methods to makecompounds of this disclosure, i.e., compounds of structures (II) and(IIA):

wherein R¹, R^(1a), R^(2a), R^(2b), R^(4a), R⁵, R⁶, R⁷, and X aredescribed above in the Brief Summary, as an isolated enantiomer or aracemic mixture thereof, or a pharmaceutically acceptable salt thereof.It is understood that in the following Reaction Schemes, combinations ofsubstituents and/or variables of the depicted structures are permissibleonly if such contributions result in stable compounds.

Generally speaking, commercially available 6-methyluracil (1) can befirst alkylated to provide 1,3-dialkyl6-methyluracil (2). Suitablealkylating agents include, for example, alkylsulfate, etc. In particularembodiments, the alkylation is methylation and the exemplary alkylatingagents include dimethylsulfate, iodomethane, etc. Thereafter,1,3-dialkyl-6-methyluracil (2) is further acylated to provide ketone (4)via, e.g., a Friedel-Crafts mechanism, in the presence of anappropriately substituted benzoyl chloride (3) and a Lewis acid catalyst(e.g., ZnCl). Ketone (4) can then be brominated to provide compound (5).

Reaction of compound (5) and an appropriately substituted (includingN-substituted) phenylenediamine (6) provides compound (7) following aring-closure step. Compound (7) may further react with aformyl-substituted furanyl or thienyl compound (8) to provide a compoundof Structure (II).

Alternatively, compound (5) may undergo a ring-closure step by firstreacting with an optionally protected phenylenediamine (9) to providecompound (10). If protected, the amino group of (10) may be deprotectedthrough conventional methods (e.g., hydrolysis) prior to reacting with aformyl-substituted furanyl or thienyl group (8). Thereafter, an R⁴ group(e.g., alkyl, nitroso) may be introduced in the form of R⁴-LG (LG beinga leaving group) to produce a ring-condensed product represented byStructure (II).

Methods of Using and Characterizing BPO and PPQ Compounds andCompositions Comprising the Compounds

As described in greater detail herein, pharmaceutical compositions areprovided, wherein the pharmaceutical compositions comprise apharmaceutically suitable excipient (i.e., a pharmaceutically acceptableexcipient or a physiologically suitable or acceptable excipient) and atleast one of the BPO or PPQ compounds of any one of the structures,substructures, and specific structures described herein, including acompound of structure I and substructures (Ia(i)), (Ib(i)), (IA), (IA1),(IB), (IB 1), (IC), (IC1), and specific compounds, and/or a compound ofstructure (II), substructure IIA, and specific compounds. The BPO andPPQ compounds described herein are capable of inhibiting CFTR activity(i.e., inhibiting, reducing, decreasing, or blocking transport ofchloride ion in the CFTR channel or pore in a statistically, clinicallyand/or biologically significant manner) in a cell and therefore may beused for treating diseases, disorders, and conditions that are treatableby inhibiting CFTR activity. Such diseases, disorder, and conditionsinclude those that result from or are related to aberrantly increasedCFTR activity. Accordingly, methods of inhibiting ion transport (e.g.,inhibiting chloride ion transport) by CFTR are provided herein.

Also as described herein, the BPO and PPQ compounds of structure I orstructure II (and substructures thereof) that are CFTR inhibitors areuseful in the treatment of a CFTR-mediated or -associated condition,i.e., any condition, disorder, or disease, that results from activity ofCFTR, such as CFTR-mediated ion transport. The condition, disorder, ordisease may result from aberrantly increased CFTR activity, particularlyaberrantly increased CFTR-mediated ion transport. These conditions,disorders, and diseases, are thus amenable to treatment by inhibitingCFTR activity, e.g., inhibiting CFTR-mediated ion, such as chloride ion,transport.

Accordingly, methods are provided for treating a disease, disorder, orcondition that is treatable by inhibiting CFTR-mediated ion transport.In certain embodiments, methods are provided for inhibiting cystformation and/or cyst enlargement, particularly kidney cyst formation orenlargement. These methods are described in greater detail below andherein.

The BPO and PPQ compounds described herein are capable of blocking orimpeding the CFTR pore or channel and inhibiting ion transport (e.g.,inhibiting chloride ion (Cl⁻) transport (also referred to as inhibitingchloride ion conductance)) by CFTR, which is located in the outer cellmembrane of a cell. Provided herein are methods of inhibiting iontransport by CFTR, which methods comprise contacting a cell that hasCFTR located in its outer membrane with any one or more of the BPO orPPQ compounds described herein, thus permitting CFTR and the compound orcompounds to interact. Interaction of a BPO or PPQ compound describedherein results in binding to CFTR, thereby inhibiting chloride iontransport.

In one embodiment, a method is provided for inhibiting (i.e.,preventing, retarding, slowing, impeding) cyst formation and/or forinhibiting (i.e., preventing, retarding, slowing, impeding) or reducingcyst enlargement, or reducing the size and/or volume of one or morecysts, which method comprises contacting (a) a cell that comprises CFTRand (b) at least one BPO or PPQ compound as described herein, underconditions and for a time sufficient for CFTR and the compound tointeract, wherein the compound inhibits ion (e.g., chloride ion)transport by CFTR, (i.e., the compound inhibits CFTR-mediated iontransport in a statistically significant, clinically significant, and/orbiologically significant manner). In particular embodiments, the cystformation or cyst enlargement that is inhibited is kidney cyst formationor kidney cyst enlargement (i.e., cyst formation or cyst enlargement inat least one kidney is inhibited).

Inhibiting kidney cyst formation and/or cyst enlargement by the BPO andPPQ compounds described herein is useful for treating a patient who hasbeen diagnosed with or who is at risk of developing polycystic kidneydisease. Accordingly, methods are provided herein for treatingpolycystic kidney disease, which methods comprise administering to asubject in need thereof (a) a pharmaceutically suitable excipient and(b) at least one of the BPO or PPQ compounds (i.e., the compounds ofstructure I and substructures (Ia(i)), (Ib(i)), (IA), (IA1), (IB),(IB1), (IC), (IC1), and specific compounds and the compounds ofstructure (II), substructures IIA, and specific compounds) as describedherein (i.e., a pharmaceutical composition as described herein). In aspecific embodiment, polycystic kidney disease is autosomal dominantpolycystic kidney disease. In another specific embodiment, polycystickidney disease is autosomal recessive polycystic kidney disease.

In another embodiment, a method for treating a disease, disorder, orcondition that is treatable by inhibiting CFTR-mediated ion transportincludes a disease, disorder, or condition that is associated withaberrantly increased CFTR-mediated ion transport. Accordingly, in aspecific embodiment, a method is provided for treating a disease,condition, or disorder associated with aberrantly increased iontransport by cystic fibrosis transmembrane conductance regulator (CFTR),wherein the method comprises administering to a subject in need thereofa pharmaceutically suitable excipient and at least one of the BPO or PPQcompounds described herein (i.e., a pharmaceutical composition asdescribed herein), wherein ion transport mediated by CFTR is inhibited.In a specific embodiment, the disease, condition, or disorder isaberrantly increased intestinal fluid secretion, which may be acuteaberrantly increased intestinal fluid secretion.

In another embodiment, the BPO and PPQ compounds of structure I andstructure II, respectively (and substructures and specific compoundsthereof) are used in the treatment of conditions associated withaberrantly increased intestinal fluid secretion, particularly acuteaberrantly increased intestinal fluid secretion, including secretorydiarrhea. Diarrhea amenable to treatment using any one of the BPO or PPQcompounds described herein can result from exposure to a variety ofagents or pathogens, including those that cause an enteropathogenicinfection. In a more specific embodiment, secretory diarrhea is causedby an enteric pathogen. Exemplary enteric pathogens include withoutlimitation, Vibrio cholerae, Escherichia coli, (particularlyenterotoxigenic E. coli (ETEC)), Shigella, Salmonella, Campylobacter(including Campylobacter jejuni), Clostridium difficile, parasites(e.g., Giardia (e.g., Giardia lamblia), Entamoeba histolytica,Cryptosporidium, Cyclospora), or diarrheal viruses (e.g., rotavirus).Secretory diarrhea resulting from increased intestinal fluid secretionmediated by CFTR may also be a disorder or sequelae associated with foodpoisoning, or exposure to a toxin including but not limited to abacterial enterotoxin such as cholera toxin (V. cholera), a E. colitoxin, a Salmonella toxin, a Campylobacter toxin, or a Shigella toxin,or any other bacterial toxin that causes aberrantly increased intestinalfluid secretion.

Other secretory diarrheas that may be treated by administering any oneor more of the BPO and PPQ compounds described herein include diarrheaassociated with or that is a sequelae of AIDS; diarrhea that is acondition related to the effects of anti-AIDS medications such asprotease inhibitors; diarrhea that is a condition of or is related toadministration of chemotherapeutic compounds; inflammatorygastrointestinal disorders, such as ulcerative colitis, inflammatorybowel disease (IBD), Crohn's disease, diverticulosis, and the like.Intestinal inflammation modulates the expression of three majormediators of intestinal salt transport and may contribute to diarrhea inulcerative colitis both by increasing transepithelial Cl⁻ secretion andby inhibiting the epithelial NaCl absorption (see, e.g., Lohi et al.,Am. J. Physiol. Gastrointest. Liver Physiol. 283:G567-75 (2002)). Thus,one or more of the BPO and PPQ compounds of structure I or structure IIand substructures thereof, and specific structures as described herein,may be administered in an amount effective to inhibit CFTR ion transportand, thus, decrease intestinal fluid secretion.

As understood by a person skilled in the medical art, the terms, “treat”and “treatment,” refer to medical management of a disease, disorder, orcondition of a subject (i.e., patient) (see, e.g., Stedman's MedicalDictionary). In general, an appropriate dose and treatment regimenprovide the BPO and/or PPQ compound in an amount sufficient to providetherapeutic and/or prophylactic benefit. Therapeutic and/or prophylacticbenefit resulting from therapeutic treatment and/or prophylactic orpreventative measures includes, for example, an improved clinicaloutcome, wherein the object is to prevent or slow or retard (lessen) anundesired physiological change or disorder, or to prevent or slow orretard (lessen) the expansion or severity of such disorder. As discussedherein, beneficial or desired clinical results from treating a subjectinclude, but are not limited to, abatement, lessening, or alleviation ofsymptoms that result from or are associated the disease, condition, ordisorder to be treated; decreased occurrence of symptoms; improvedquality of life; longer disease-free status (i.e., decreasing thelikelihood or the propensity that a subject will present symptoms on thebasis of which a diagnosis of a disease is made); diminishment of extentof disease; stabilized (i.e., not worsening) state of disease; delay orslowing of disease progression; amelioration or palliation of thedisease state; and remission (whether partial or total), whetherdetectable or undetectable; and/or overall survival. “Treatment” canalso mean prolonging survival when compared to expected survival if asubject were not receiving treatment. Subjects in need of treatmentinclude those who already have the condition or disorder as well assubjects prone to have or at risk of developing the disease, condition,or disorder, and those in which the disease, condition, or disorder isto be prevented (i.e., decreasing the likelihood of occurrence of thedisease, disorder, or condition).

Other embodiments provided herein include use of at least one of the BPOand PPQ compounds of structure I, structure II, substructures, andspecific structures as described herein for treating any one of thediseases or disorders described herein (e.g., polycystic kidney disease,aberrantly increased intestinal fluid secretion, secretory diarrhea)that is treatable by inhibiting ion transport (e.g., chloride iontransport) by CFTR. In one embodiment, a use is provided for thepreparation of a medicament for treating any one of the diseases,conditions or disorders described herein (e.g., polycystic kidneydisease, aberrantly increased intestinal fluid secretion, secretorydiarrhea) that is treatable by inhibiting ion transport (e.g., chlorideion transport) by CFTR.

In other embodiments, methods are provided for treating a disease,disorder, or condition described herein (including but not limited toPCKD, secretory diarrhea or other condition associated with aberrantlyincreased intestinal fluid secretion). Such methods compriseadministering the compound or a pharmaceutical composition thatcomprises at least one BPO or PPQ compound and a pharmaceuticallysuitable (i.e., pharmaceutically acceptable, or physiologically suitableor acceptable) excipient in combination with, either in the samecomposition or in a separate (or second) composition, at least onethiazolidinone compound and/or at least one glycine hydrazide compoundthat inhibit CFTR-mediated ion transport (see, e.g., U.S. Pat. Nos.7,235,573; 7,414,037; U.S. Patent Application Publication No.2008/0064666; International Patent Application Publication No. WO2008/079897; U.S. Patent Application Publication No. US2009/0253799;International Patent Application No. PCT/US2009/038292, which are herebyincorporated by reference in their entireties) for treating any one ofthe diseases or disorders described herein that is treatable byinhibiting ion transport (e.g., chloride ion transport) by CFTR. When afirst composition comprising at least one BPO or PPQ compound describedherein and a second composition comprising at least one thiazolidinonecompound and/or at least one glycine hydrazide compound is administeredto a subject in need thereof, the first composition may be administeredprior to, concurrently with, or subsequent to administration of thesecond composition.

In particular embodiments of the methods described herein, the subjectis a human or non-human animal. A subject in need of the treatmentsdescribed herein may exhibit symptoms or sequelae of a disease,disorder, or condition described herein or may be at risk of developingthe disease, disorder, or condition. Non-human animals that may betreated include mammals, for example, non-human primates (e.g., monkey,chimpanzee, gorilla, and the like), rodents (e.g., rats, mice, gerbils,hamsters, ferrets, rabbits), lagomorphs, swine (e.g., pig, miniaturepig), equine, canine, feline, bovine, and other domestic, farm, and zooanimals.

In another embodiment, a method is provided for inhibiting ion transportby a cystic fibrosis transmembrane conductance regulator (CFTR)comprising contacting (a) a cell that comprises CFTR and (b) at leastone of the compounds of structure I or structure II, substructures, andspecific structures described herein, under conditions and for a timesufficient that permit CFTR and the compound to interact, therebyinhibiting ion transport (e.g., chloride ion transport) by CFTR, thatis, inhibiting CFTR-mediated ion transport.

In another embodiment, a method is provided for treating secretorydiarrhea comprising administering to a subject in need thereof apharmaceutically acceptable excipient and at least one of the compoundsof structure I or structure II, substructures, and specific structuresdescribed herein (i.e., a pharmaceutical composition as describedherein). In a particular embodiment, the subject is a human or non-humananimal.

The pharmaceutical compositions and methods of using the PPQ compoundsand compositions comprising these compounds are described in greaterdetail herein.

Methods for Characterizing and Using the BPO and PPQ Compounds

Also provided herein are methods that are useful, for example, forcharacterizing the potency of BPO and PPQ compounds (and derivatives andanalogs thereof) to inhibit CFTR-mediated ion transport (particularlyCFTR-mediated chloride ion transport); for monitoring the level (i.e.,for example, concentration level, mass level, or IC₅₀ level) of a BPO orPPQ compound that has been administered to a subject; and examiningdisease pathogenesis in cystic fibrosis by blocking or inhibiting CFTRfunction as models for cystic fibrosis disease, such as in ex vivotissues (e.g., human tissues) and in animals.

In certain embodiments, these methods may be performed in vitro, such aswith using a biological sample as described herein that comprises, forexample, cells obtained from a tissue, body fluid, or culture-adaptedcell line, or other biological source as described in detail hereinbelow. The step of contacting refers to combining, mixing, or in somemanner familiar to persons skilled in the art that permits the compoundand the cell to interact such that any effect of the compound on CFTRactivity (e.g., the capability of a BPO or PPQ compound to inhibit CFTRion transport or the level to which the compound inhibits CFTR iontransport) can be measured according to methods described herein androutinely practiced in the art. Methods described herein for inhibitingion transport by CFTR are understood to be performed under conditionsand for a time sufficient that permit the CFTR and the compound tointeract. Additional BPO and PPQ compounds may be identified and/orcharacterized by such a method of inhibiting ion transport by CFTR,performed with isolated cells in vitro. Conditions for a particularassay include temperature, buffers (including salts, cations, media),and other components that maintain the integrity of the cell and thecompound, which a person skilled in the art will be familiar and/orwhich can be readily determined. A person skilled in the art alsoreadily appreciates that appropriate controls can be designed andincluded when performing the in vitro methods and in vivo methodsdescribed herein.

In secretory epithelia, fluid secretion occurs by primary chloride exitacross the cell apical membrane, which secondarily drivestransepithelial sodium and water secretion (see, e.g., Barrett et al.,Annu. Rev. Physiol. 62:535-72 (2000)). In renal cells, lumenal fluidaccumulation causes progressive cyst expansion directly by net waterinflux into the cyst lumen, and indirectly by stretching cyst wallepithelial cells to promote their division and thinning (Ye et al., NEngl. J. Med. 329:310-13 (1993); Sullivan et al., Physiol. Rev.78:1165-91 (1998); Tanner et al., J. Am. Soc. Nephrol. 6:1230-41(1995)). Without wishing to be bound by any particular theory, CFTRinhibition interferes with fluid secretion at the apical chloride exitstep.

Methods for characterizing the compounds described herein, fordetermining an effective concentration to achieve a therapeutic benefit,for monitoring the level of a BPO or PPQ compound in a biologicalsample, and for other purposes as described herein and apparent to aperson skilled in the art, may be performed using techniques andprocedures described herein and routinely practiced by a person skilledin the art. Exemplary methods include, but are not limited to,fluorescence cell-based assays of CFTR inhibition (see, e.g., Galiettaet al., J. Physiol. 281:C1734-C1742 (2001); Ma et al., J. Clin. Invest.110:1651-58 (2002)), short circuit apical chloride ion currentmeasurements and patch-clamp analysis (see, e.g., Muanprasat et al., J.Gen. Physiol. 124:125-37 (2004); Ma et al., J. Clin. Invest. (2002),supra; Sonawane et al., FASEB J. 20:130-32 (2006); see also, e.g.,Carmeliet, Verh. K Acad. Geneeskd. Belg. 55:5-26 (1993); Hamill et al.,Pflugers Arch. 391:85-100 (1981)).

Methods that may be used to characterize a BPO or a PPQ compound,including those described herein, and to determine effectiveness of thecompound for reducing, inhibiting, or preventing cyst enlargement and/orpreventing or inhibiting cyst formation, and which compound is thereforeuseful for treating a subject who has or who is at risk of developingPKD, include methods described in the art and herein. The PPQ compoundsmay be analyzed in models using embryonic or neonatal kidney organcultures, for example (see, e.g., Yang et al., J. Am. Soc. Nephrol.19:1300-1310 (2008); Magenheimer et al., J. Am. Soc. Nephrol. 17:3424-37(2006); Tradtrantip et al., J. Med. Chem. 52:6447-55 (2009)). Withoutwishing to be bound by any particular theory, certain scientificobservations support use of CFTR inhibitors to slow cyst growth inautosomal dominant PKD (ADPKD): (a) CFTR is expressed strongly inepithelial cells lining cysts in ADPKD; (b) cystic fibrosis(CFTR-deficient) mice are resistant to cyst formation; (c) CFTRinhibitors block cyst formation in cell/organ culture and in vivomodels; and (d) in some families affected with ADPKD and cysticfibrosis, individuals with both ADPKD and CF have less severe renaldisease than those with ADPKD only (see, e.g., O'Sullivan et al., Am. J.Kidney Dis. 32:976-983 (1998); Cotton et al., Am. J. Kidney Dis.32:1081-1083 (1998); Xu et al., J. Nephrol. 19:529-34 (2006)). Intactkidney models to study cystogenesis are useful for recapitulating nativekidney anatomy and cellular phenotype (see, e.g., Magenheimer et al., J.Am. Soc. Nephrol. 17:3424-3437 (2006)).

An additional example of a cell culture model for determining whether acompound inhibits cyst formation or enlargement includes an MDCK cell(Madin-Darby Canine Kidney Epithelial Cell) model of PKD (Li et al.,Kidney Int 66:1926-38 (2004); see also, e.g., Neufeld et al., KidneyInt. 41:1222-36 (1992); Mangoo-Karim et al., Proc. Natl. Acad. Sci. USA86:6007-6011 (1989); Mangoo-Karim et al., FASEB J. 3:2629-32 (1989);Grantham et al., Trans. Assoc. Am. Physic. 102:158-62 (1989); Mohamed etal., Biochem. J. 322: 259-65 (1997)). See also, e.g., Murcia et al.,Kidney Int. 55:1187-97 (1999); Igarishi et al., J. Am. Soc. Nephrol.13:2384-88 (2002)). Accordingly, provided herein are methods foridentifying and/or characterizing BPO and PPQ compounds of structure Ior structure II (and substructures and specific structures thereof) bydetermining the capability of the compound to inhibit cyst enlargementor prevent or inhibit cyst formation in an in vitro cell culture model.

The MDCK cell line may also be used in methods and techniques fordetermining that a compound lacks cytotoxicity, for example, byevaluating cell viability (e.g., by any one of numerous cell stainingmethods and microscopy methods routinely practiced in the art), cellproliferation (e.g., by determining the level of incorporation ofnucleotide analogs and other methods for measuring division of cells),and/or apoptosis by using any one of a number of techniques and methodsknown in the art and described herein. Other methods for determining orquantifying the capability of a compound described herein to inhibit orreverse cyst enlargement or expansion and/or to inhibit or prevent cystformation and/or to reduce the number of cysts formed include anembryonic kidney organ culture model, which is practiced in the art anddescribed herein (see, e.g., Magenheimer et al., J. Am. Soc. Nephrol.17: 3424-37 (2006); Steenhard et al., J. Am. Soc. Nephrol. 16:1623-1631(2005); Yang et al., J. Am. Soc. Nephrol. 19:1300-1310 (2008)). In suchan embryonic kidney culture model, organotypic growth anddifferentiation of renal tissue can be monitored in defined media in theabsence of any effect or influence by circulating hormones andglomerular filtration (Magenheimer et al., supra; Gupta et al., KidneyInt 63:365-376 (2003)). In metanephric organ culture, the early mousekidney tubule has an intrinsic capacity to secrete fluid by aCFTR-dependent mechanism in response to cAMP (Magenheimer et al.,supra).

Persons skilled in the art may also use animal models to characterize aBPO or PPQ compound, including those described herein, and to determineeffectiveness of the compound for reducing, inhibiting, reversing, orpreventing cyst enlargement and/or preventing or inhibiting cystformation thereby reducing the number of cysts formed, and to determinethe usefulness of such compounds for treating a subject who has or whois at risk of developing MD. By way of example, Pkd1^(flox) mice andKsp-Cre transgenic mice in a C57BL/6 background may be generated asdescribed and practiced in the art (see, e.g., Shibazaki et al., J. Am.Soc. Nephrol. 13:10-11 (2004) (abstract); Shao et al., J. Am. Soc.Nephrol. 13:1837-46 (2002)). Ksp-Cre mice express Cre recombinase underthe control of the Ksp-cadherin promoter (see, e.g., Shao et al.,supra). Pkd1^(flox/−); Ksp-Cre mice may be generated by cross-breedingPkd1^(flox/flox) mice with Pkd1^(+/−):Ksp-Cre mice. The effect of a testcompound may be determined by quantifying cyst size and growth inmetanephroi and kidney sections, histological analyses of tissues andcells, and delay or prevention of renal failure and death (see, e.g.,Tradtrantip et al., J. Med. Chem., supra; Shibazaki et al., supra).

The BPO and PPQ compounds may also be analyzed in animal models that areart-accepted animal models for increased intestinal fluid secretion. Byway of example, a closed intestinal loop model of cholera, sucklingmouse model of cholera, and in vivo imaging of gastrointestinal transitmay be used for characterizing the BPO and PPQ compounds describedherein (see, e.g., Takeda et al., Infect. Immun. 19:752-54 (1978); seealso, e.g., Spira et al., Infect. Immun. 32:739-747 (1981)). Animalmodels of secretory diarrheas have been useful for determining efficacyof thiazolidinone (see, e.g., Thiagarajah et al., Gastroenterology.126:511-19 (2004)) and glycine hydrazide CFTR inhibitors (see, e.g.,Sonawane et al., Chem. Biol. 15:718-28 (2008); Sonawane et al.,Gastroenterology 132:1234-44 (2007)) and may be employed for testing theBPO and PPQ compounds described herein.

The BPO and PPQ compounds described herein may also be useful forestablishing animal models that may be used as cystic fibrosis models.The CF phenotype (in the absence of the CF genotype) may be establishedby administering any one or more of the BPO and PPQ compounds describedherein. CFTR inhibitors are also useful to create the CF phenotype in exvivo human and animal tissues, as has been done, for example, in studiesof the role of CFTR in airway submucosal gland fluid secretion (see,e.g., Thiagarajah et al., FASEB J. 18:875-77 (2004)). Finally, thoughmouse, pig and ferret models of CFTR gene deletion exist,pharmacological creation of the CFTR phenotype in animals by CFTRinhibitors might provide complementary data on CFTR function in theabsence of compensatory phenomena that can occur in transgenic animalmodels.

Methods of inhibiting CFTR-mediated ion transport include in vitromethods that comprise contacting a cell with any one or more of the BPOand/or PPQ compounds of structure I or structure II (and substructuresand specific structures thereof) as described herein, under conditionsand for a time sufficient for CFTR present in the outer membrane of thecell and the compound to interact. Cells (or cell lines) that may beused in such methods are cells that express CFTR and have channels orpores formed by CFTR in the cell membrane. Exemplary cells and celllines include without limitation a Fischer rat thyroid (FRT) cell(including a FRT cell that co-expresses human or other animal wildtypeCFTR and the halide indicator YFP-H148Q or other comparable yellowfluorescent protein); a cultured human bronchial epithelial cell; and agastrointestinal cell (such as T84 human intestinal epithelial cells))that comprises CFTR in the outer membrane of the cell. Such methods areuseful for identifying analogs of the BPO and PPQ compounds (i.e.,species of structure I or structure II, including species ofsubstructures described herein) and for characterizing the BPO and PPQcompounds described herein.

In certain embodiments, the cell is contacted in an in vitro assay, andthe cell may be obtained from a subject or from a biological sample. Abiological sample may be a blood sample (from which serum or plasma maybe prepared and cells isolated), biopsy specimen, body fluids (e.g.,urine, lung lavage, ascites, mucosal washings, synovial fluid), bonemarrow, lymph nodes, tissue explant (e.g., kidney cells), organ culture(e.g., kidney), or any other tissue or cell preparation from a subjector a biological source. A sample may further refer to a tissue or cellpreparation in which the morphological integrity or physical state hasbeen disrupted, for example, by dissection, dissociation,solubilization, fractionation, homogenization, biochemical or chemicalextraction, pulverization, lyophilization, sonication, or any othermeans for processing a sample derived from a subject or biologicalsource. The subject or biological source may be a human or non-humananimal, a primary cell culture (e.g., a primary kidney cell culture orprimary intestinal epithelial cell culture), or culture adapted cellline, including but not limited to, genetically engineered cell linesthat may contain chromosomally integrated or episomal recombinantnucleic acid sequences, immortalized or immortalizable cell lines,somatic cell hybrid cell lines, differentiated or differentiatable celllines, transformed cell lines, and the like.

The BPO and PPQ compounds described herein may also be used in any oneor more of the assays described above and herein for in vitro screeningmethods. By way of non-limiting examples, the compounds may be used ascontrols for comparing the potency, polarity, solubility, or otherproperties of compounds in compound libraries when screening assays areperformed to identify and characterize compounds that inhibit CFTRactivity.

Pharmaceutical Compositions and Methods of Using PharmaceuticalCompositions

Also provided herein are pharmaceutical compositions comprising any oneor more of the BPO and PPQ compounds of structure I or structure II (andsubstructures and specific structures thereof). The compounds describedherein may be formulated in a pharmaceutical composition for use intreatment or preventive (or prophylactic) treatment (e.g., reducing thelikelihood of occurrence of cyst formation), of polycystic kidneydisease (PKD), which includes autosomal dominant PKD (ADPKD) andautosomal recessive PKD (ARPKD). In other embodiments, a pharmaceuticalcomposition comprising at least one BPO or PPQ compound may beformulated for use in treatment or preventive treatment (i.e., forreducing the likelihood of occurrence) of a disease, condition, ordisorder manifested by increased intestinal fluid secretion, such assecretory diarrhea.

In pharmaceutical dosage forms, any one or more of the BPO and PPQcompounds of structure I, substructures, and specific structures orstructure II, substructures, and specific structures described hereinmay be administered in the form of a pharmaceutically acceptablederivative, such as a salt, or they may also be used alone or inappropriate association, as well as in combination, with otherpharmaceutically active compounds. The methods and excipients describedherein are merely exemplary and are in no way limiting.

Optimal doses may generally be determined using experimental modelsand/or clinical trials. The optimal dose may depend upon the body mass,weight, or blood volume of the subject. In general, the amount of a BPOor PPQ compound of structure I or structure II (and substructures andspecific structures thereof) described herein, that is present in adose, ranges from about 0.01 μg to about 1000 μg per kg weight of thehost. The use of the minimum dose that is sufficient to provideeffective therapy is usually preferred. Subjects may generally bemonitored for therapeutic effectiveness using assays suitable for thecondition being treated or prevented, which assays will be familiar tothose having ordinary skill in the art and are described herein. Thelevel of a compound that is administered to a subject may be monitoredby determining the level of the compound in a biological fluid, forexample, in the blood, blood fraction (e.g., serum), and/or in theurine, and/or other biological sample from the subject. Any methodpracticed in the art to detect the compound may be used to measure thelevel of compound during the course of a therapeutic regimen.

The dose of a composition comprising at least one of the BPO or PPQcompounds described herein for treating PCKD may depend upon thesubject's condition, that is, stage of the disease, renal function,severity of symptoms caused by enlarged cysts, general health status, aswell as age, gender, and weight, and other factors apparent to a personskilled in the medical art. Similarly, the dose of the BPO or PPQcompound for treating a disease or disorder associated with aberrantlyincreased CFTR function, including but not limited to intestinal fluidsecretion, secretory diarrhea, such as a toxin-induced diarrhea, orsecretory diarrhea associated with or a sequelae of an enteropathogenicinfection, Traveler's diarrhea, ulcerative colitis, irritable bowelsyndrome (IBS), AIDS, chemotherapy and other diseases or conditionsdescribed herein may be determined according to parameters understood bya person skilled in the medical art. Accordingly, the appropriate dosemay depend upon the subject's condition, that is, stage of the disease,general health status, as well as age, gender, and weight, and otherfactors considered by a person skilled in the medical art.

Pharmaceutical compositions may be administered in a manner appropriateto the disease or disorder to be treated as determined by personsskilled in the medical arts. An appropriate dose and a suitable durationand frequency of administration will be determined by such factors asthe condition of the patient, the type and severity of the patient'sdisease, the particular form of the active ingredient, and the method ofadministration. In general, an appropriate dose (or effective dose) andtreatment regimen provides the composition(s) comprising at least oneBPO or PPQ compound (as described herein) in an amount sufficient toprovide therapeutic and/or prophylactic benefit (for example, animproved clinical outcome, such as more frequent complete or partialremissions, or longer disease-free and/or overall survival, or alessening of symptom severity or other benefit as described in detailabove). When a subject is treated for aberrantly increased intestinalfluid secretion, clinical assessment of the level of dehydration and/orelectrolyte imbalance may be performed to determine the level ofeffectiveness of a compound and whether dose or other administrationparameters (such as frequency of administration or route ofadministration) should be adjusted.

Polycystic kidney disease (PKD) (or PCKD) and polycystic renal diseaseare used interchangeably herein, and refer to a group of disorderscharacterized by a large number of cysts distributed throughout enlargedkidneys. The resultant cyst development leads to impairment of kidneyfunction and can eventually cause kidney failure. PKD includes autosomaldominant polycystic kidney disease (ADPKD) and recessive autosomalrecessive polycystic kidney disease (ARPKD), in all stages ofdevelopment, regardless of the underlying etiology or cause.Effectiveness of a treatment for PKD may be monitored by one or more ofseveral methods practiced in the medical art including, for example, bymonitoring renal function by standard measurements, and by radiologicinvestigations that are performed with ultrasounds, computerizedtomography (CT), or magnetic resonance imaging, which are useful forevaluating renal cyst morphology and volume and estimating the amount ofresidual renal parenchyma.

To evaluate and to monitor the effectiveness of any one of the BPO orPPQ compounds described herein to treat PKD or a related disease orcondition, one or more of several clinical assay methods may beperformed that are familiar to a person skilled in the clinical art. Forexample, a clinical method called a urea clearance test may beperformed. A blood sample is obtained from a subject to whom thecompound is being administered so that the amount of urea in thebloodstream can be determined. In addition, a first urine sample iscollected from the subject and at least one hour later, a second urinesample is collected. The amount of urea quantified in the urineindicates the amount of urea that is filtered, or cleared by the kidneysinto the urine. Another clinical assay method measures urine osmolality(i.e., the amount of dissolved solute particles in the urine). Inabilityof the kidneys to concentrate the urine in response to restricted fluidintake, or to dilute the urine in response to increased fluid intakeduring osmolality testing may indicate decreased kidney function.

Urea is a by-product of protein metabolism and is formed in the liver.Urea is then filtered from the blood and excreted in the urine by thekidneys. The BUN (blood urea nitrogen) test measures the amount ofnitrogen contained in the urea. High BUN levels may indicate kidneydysfunction, but because blood urea nitrogen is also affected by proteinintake and liver function, the test is usually performed in conjunctionwith determination of blood creatinine, which is considered a morespecific indicator of kidney function. Low clearance values forcreatinine and urea indicate diminished ability of the kidneys to filterthese waste products from the blood and excrete them in the urine. Asclearance levels decrease, blood levels of creatinine and urea nitrogenincrease. An abnormally elevated blood creatinine, a more specific andsensitive indicator of kidney disease than the BUN, is diagnostic ofimpaired kidney function.

The pharmaceutical compositions described herein that comprise at leastone BPO or PPQ compound may be administered to a subject in need by anyone of several routes that effectively deliver an effective amount ofthe compound. Such administrative routes include, for example, oral,parenteral, enteral, rectal, intranasal, buccal, sublingual,intramuscular, and transdermal. By way of example, at least one or moreof the compounds may be administered to a mucosal surface of thegastrointestinal tract (e.g., by an enteral route, which includesadministration directly to the tract via a tube inserted into the nose,stomach, or small intestine) or to a mucosal surface of the oral ornasal cavities, or (e.g., intranasal, buccal, sublingual, and the like).These administrative methods and additional methods are discussed ingreater detail herein.

A pharmaceutical composition may be a sterile aqueous or non-aqueoussolution, suspension or emulsion, which additionally comprises aphysiologically acceptable excipient (pharmaceutically acceptable orsuitable excipient or carrier) (i.e., a non-toxic material that does notinterfere with the activity of the active ingredient). Such compositionsmay be in the form of a solid, liquid, or gas (aerosol). Alternatively,compositions described herein may be formulated as a lyophilizate, orcompounds may be encapsulated within liposomes using technology known inthe art. Pharmaceutical compositions may also contain other components,which may be biologically active or inactive. Such components include,but are not limited to, buffers (e.g., neutral buffered saline orphosphate buffered saline), carbohydrates (e.g., glucose, mannose,sucrose or dextrans), mannitol, proteins, polypeptides or amino acidssuch as glycine, antioxidants, chelating agents such as EDTA orglutathione, stabilizers, dyes, flavoring agents, and suspending agentsand/or preservatives.

Any suitable excipient or carrier known to those of ordinary skill inthe art for use in pharmaceutical compositions may be employed in thecompositions described herein. Excipients for therapeutic use are wellknown, and are described, for example, in Remington: The Science andPractice of Pharmacy (Gennaro, 21^(st) Ed. Mack Pub. Co., Easton, Pa.(2005)). In general, the type of excipient is selected based on the modeof administration, as well as the chemical composition of the activeingredient(s). Pharmaceutical compositions may be formulated for anyappropriate manner of administration, including, for example, topical,oral, nasal, intrathecal, rectal, vaginal, intraocular, subconjunctival,sublingual or parenteral administration, including subcutaneous,intravenous, intramuscular, intrasternal, intracavernous, intrameatal orintraurethral injection or infusion. For parenteral administration, thecarrier preferably comprises water, saline, alcohol, a fat, a wax or abuffer. For oral administration, any of the above excipients or a solidexcipient or carrier, such as mannitol, lactose, starch, magnesiumstearate, sodium saccharine, talcum, cellulose, kaolin, glycerin, starchdextrins, sodium alginate, carboxymethylcellulose, ethyl cellulose,glucose, sucrose and/or magnesium carbonate, may be employed.

A pharmaceutical composition (e.g., for oral administration or deliveryby injection) may be in the form of a liquid. A liquid pharmaceuticalcomposition may include, for example, one or more of the following: asterile diluent such as water for injection, saline solution, preferablyphysiological saline, Ringer's solution, isotonic sodium chloride, fixedoils that may serve as the solvent or suspending medium, polyethyleneglycols, glycerin, propylene glycol or other solvents; antibacterialagents; antioxidants; chelating agents; buffers and agents for theadjustment of tonicity such as sodium chloride or dextrose. A parenteralpreparation can be enclosed in ampoules, disposable syringes or multipledose vials made of glass or plastic. The use of physiological saline ispreferred, and an injectable pharmaceutical composition is preferablysterile.

A composition comprising any one of the compounds of structure (I) orstructure (II), substructures, and specific structures described hereinmay be formulated for sustained or slow release. Such compositions maygenerally be prepared using well known technology and administered by,for example, oral, rectal or subcutaneous implantation, or byimplantation at the desired target site. Sustained-release formulationsmay contain the compound dispersed in a carrier matrix and/or containedwithin a reservoir surrounded by a rate controlling membrane. Excipientsfor use within such formulations are biocompatible, and may also bebiodegradable; preferably the formulation provides a relatively constantlevel of active component release. The amount of active compoundcontained within a sustained release formulation depends upon the siteof implantation, the rate and expected duration of release, and thenature of the condition to be treated or prevented.

For oral formulations, a BPO or PPQ compound of structure (I) andsubstructures and specific structures or of structure (II) andsubstructures and specific structures described herein can be used aloneor in combination with appropriate additives to make tablets, powders,granules or capsules, for example, with conventional additives, such aslactose, mannitol, corn starch or potato starch; with binders, such asstarch, gelatin, natural sugars such as glucose or beta-lactose, cornsweeteners, natural and synthetic gums such as acacia, tragacanth, orsodium alginate, carboxymethylcellulose, polyethylene glycol, waxes,crystalline cellulose, cellulose derivatives, and acacia; withdisintegrators, such as corn starch, potato starch or sodiumcarboxymethylcellulose, methyl cellulose, agar, bentonite, or xanthangum; with lubricants, such as talc, sodium oleate, magnesium stearatesodium stearate, sodium benzoate, sodium acetate, or sodium chloride;and if desired, with diluents, buffering agents, moistening agents,preservatives, coloring agents, and flavoring agents. The compounds maybe formulated with a buffering agent to provide for protection of thecompound from low pH of the gastric environment and/or an entericcoating. A BPO or PPQ compound may be formulated for oral delivery witha flavoring agent, e.g., in a liquid, solid or semi-solid formulationand/or with an enteric coating.

Oral formulations may be provided as gelatin capsules, which may containthe active compound along with powdered carriers, such as lactose,starch, cellulose derivatives, magnesium stearate, stearic acid, and thelike. Similar carriers and diluents may be used to make compressedtablets. Tablets and capsules can be manufactured as sustained releaseproducts to provide for continuous release of active ingredients over aperiod of time. Compressed tablets can be sugar coated or film coated tomask any unpleasant taste and protect the tablet from the atmosphere, orenteric coated for selective disintegration in the gastrointestinaltract. Liquid dosage forms for oral administration may contain coloringand/or flavoring agents to increase acceptance of the compound by thesubject.

The BPO and PPQ compounds of structure I or structure II (andsubstructures and specific structures thereof) described herein can bemade into suppositories by mixing with a variety of bases such asemulsifying bases or water-soluble bases. These compounds may beadministered rectally via a suppository. The suppository can includevehicles such as cocoa butter, carbowaxes and polyethylene glycols,which melt at body temperature, yet are solidified at room temperature.

The BPO and PPQ compounds of structure I or structure II (andsubstructures and specific structures thereof) described herein may beused in aerosol formulation to be administered via inhalation. Thecompounds may be formulated into pressurized acceptable propellants suchas dichlorodifluoromethane, propane, nitrogen and the like.

Any one or more of the BPO and PPQ compounds of structure I or structureII (and substructures and specific structures thereof) described hereinmay be administered topically (e.g., by transdermal administration).Topical formulations may be in the form of a transdermal patch,ointment, paste, lotion, cream, gel, and the like. Topical formulationsmay include one or more of a penetrating agent, thickener, diluent,emulsifier, dispersing aid, or binder. When a BPO or PPQ compound isformulated for transdermal delivery, the compound may be formulated withor for use with a penetration enhancer. Penetration enhancers, whichinclude chemical penetration enhancers and physical penetrationenhancers, facilitate delivery of the compound through the skin, and mayalso be referred to as “permeation enhancers” interchangeably. Physicalpenetration enhancers include, for example, electrophoretic techniquessuch as iontophoresis, use of ultrasound (or “phonophoresis”), and thelike. Chemical penetration enhancers are agents administered eitherprior to, with, or immediately following compound administration, whichincrease the permeability of the skin, particularly the stratum corneum,to provide for enhanced penetration of the drug through the skin.Additional chemical and physical penetration enhancers are described in,for example, Transdermal Delivery of Drugs, A. F. Kydonieus (ED) 1987CRL Press; Percutaneous Penetration Enhancers, eds. Smith et al. (CRCPress, 1995); Lenneruas et al., J. Pharm. Pharmacol. 2002;54(4):499-508; Karande et al., Pharm. Res. 2002; 19(5):655-60; Vaddi etal., Int. J. Pharm. 2002 July; 91(7):1639-51; Ventura et al., J. DrugTarget 2001; 9(5):379-93; Shokri et al., Int. J. Pharm. 2001;228(1-2):99-107; Suzuki et al., Biol. Pharm. Bull. 2001; 24(6):698-700;Alberti et al., J. Control Release 2001; 71(3):319-27; Goldstein et al.,Urology 2001; 57(2):301-5; Kiijavainen et al., Eur. J. Pharm. Sci. 2000;10(2):97-102; and Tenjarla et al., Int. J. Pharm. 1999; 192(2):147-58.

When a BPO or PPQ compound of structure I or structure II (andsubstructures and specific structures thereof) is formulated with achemical penetration enhancer, the penetration enhancer is selected forcompatibility with the compound, and is present in an amount sufficientto facilitate delivery of the compound through skin of a subject, e.g.,for delivery of the compound to the systemic circulation. The BPO andPPQ compounds of structure I or structure II (and substructures andspecific structures thereof) may be provided in a drug delivery patch,e.g., a transmucosal or transdermal patch, and can be formulated with apenetration enhancer. The patch generally includes a backing layer,which is impermeable to the compound and other formulation components, amatrix in contact with one side of the backing layer, which matrixprovides for sustained release, which may be controlled release, of thecompound, and an adhesive layer, which is on the same side of thebacking layer as the matrix. The matrix can be selected as is suitablefor the route of administration, and can be, for example, a polymeric orhydrogel matrix.

In one embodiment, the BPO and PPQ compounds of structure I or structureII (and substructures and specific structures thereof) are delivered tothe gastrointestinal tract of the subject to provide for decreased fluidsecretion. Suitable formulations for this embodiment include anyformulation that provides for delivery of the compound to thegastrointestinal surface, particularly an intestinal tract surface.

For use in the methods described herein, one or more of the BPO and PPQcompounds of structure I or structure II (and substructures and specificstructures thereof) described herein may be formulated with otherpharmaceutically active agents or compounds, including otherCFTR-inhibiting agents and compounds or agents and compounds that blockintestinal chloride channels (e.g., a glycine hydrazide compound orthiazolidinone compound as discussed herein). Similarly, one or more ofthe BPO and PPQ compounds of structure I or structure II (andsubstructures and specific structures thereof) described herein may beformulated with other pharmaceutically active agents or compounds,including other CFTR-inhibiting agents and compounds, or other agentsand compounds that are administered to a subject for treating PKD.

Kits with unit doses of a BPO or PPQ compound of structure I orstructure II (and substructures and specific structures thereof)described herein, usually in oral or injectable doses, are provided.Such kits may include a container containing the unit dose, aninformational package insert describing the use and attendant benefitsof the drugs in treating pathological condition of interest, andoptionally an appliance or device for delivery of the composition.

Also provided herein are methods of manufacturing the pharmaceuticalcompositions described herein that comprise at least one of the BPO andPPQ compounds of structure I or structure II, and substructures andspecific structures thereof, as described herein. In one embodiment, themethod of manufacture comprises synthesis of the compound. Synthesis ofone of more of the compounds described herein may be performed accordingto methods described herein and practiced in the art. In another methodof manufacture, the method comprises comprise formulating (i.e.,combining, mixing) at least one of the compounds disclosed herein with apharmaceutically suitable excipient. These methods are performed underconditions that permit formulation and/or maintenance of the desiredstate (i.e., liquid or solid, for example) of each of the compound andexcipient. A method of manufacture may comprise one or more of the stepsof synthesizing the at least one compound, formulating the compound withat least one pharmaceutically suitable excipient to form apharmaceutical composition, and dispensing the formulated pharmaceuticalcomposition in an appropriate vessel (i.e., a vessel appropriate forstorage and/or distribution of the pharmaceutical composition).

Other embodiments and uses will be apparent to one skilled in the art inlight of the present disclosures. The following examples are providedmerely as illustrative of various embodiments and shall not be construedto limit the claims in any way.

EXAMPLES Example 1 Overview of Chemistry Procedures

Synthesis Overview: NMR spectra (¹H at 600 MHz; ¹³C at 150 MHz) wereobtained in methylene chloride (CD₂Cl₂), chloroform (CDCl₃),acetonitrile (CD₃CN) or dimethyl sulfoxide (DMSO-d₆) using a 600-MHzVarian Spectrometer. Chemical shifts are expressed in parts per millionrelative to the solvent. NMR spectra for PPQ and BPO compounds wereacquired at −20° C. due to excessive broadening of the 11-phenyl protonsat ambient temperature. NMR spectra for the intermediates were obtainedat ambient temperature. Mass spectrometry was done using a Waters LC/MSinstrument with MS: electrospray (+) ionization, mass ranging from 100to 900 Da, 20-V cone voltage; LC: Xterra MS C₁₈ column (2.1 mm×50 mm×3.5μm), 0.2 mL/min water/acetonitrile (containing 0.1% TFA). Purity wasjudged by the peak area percentage of the UV absorbance signal. Compoundpurities by RP-HPLC were >98%. Flash chromatography was done using EMsilica gel (230-400 mesh), and thin-layer chromatography was done on EMDsilica gel 60 F254 plates (Darmstadt, Germany). Melting points areuncorrected.

FIG. 2 presents Scheme 1, illustrating synthesis of dihydroquinoxolinePPQ compounds, and FIG. 3 presents Scheme 2, illustrating synthesis ofbenzoxazine BPO compounds.

Efforts were undertaken to improve methods for synthesizing PPQ-102because the original synthesis method developed had low yield (see,e.g., Tradtrantip et al., J. Med. Chem. 52:6447-55 (2009); Int'l PatentAppl. Publication No. WO 2011/019737). As shown in Scheme1,6-methyluracil 1 was exhaustively alkylated using dimethyl sulfate togive 1,3,6-trimethyluracil 2 in 98% yield. 1,3,6-Trimethyluracil 2 wassubject to Friedel-Crafts acylation utilizing benzoyl chloride andanhydrous zinc chloride to give ketone 3a in 66% yield. Bromination ofketone 3a gave 4a in quantitative yield. At the first point ofdiversification, 4a was reacted with substituted 1,2-phenylenediamines(2 eq) to give pyrroles 5a-c in 97% (R²═H), 89% (R²═NO₂), and 83%(R³=Me) yields. Pyrroles 5a-c were condensed with the appropriatelysubstituted furfural or thiophene carbaldehyde using catalytic acid togive PPQ-(5-15, 23) with yields of 57-98%. PPQ-102 was obtained on agram scale in 83% yield. Amide analogs were synthesized from PPQ-102 andacid halides or anhydrides to give PPQ-(1-3), in 73%, 80% and 79%yields, respectively. The nitrosamine PPQ-4 was synthesized from PPQ-102and t-butyl nitrite in 79% yield.

Scheme 2 shows the synthesis of benzoxazine BPO compounds. Ketone 3d(R¹=Me) was synthesized from 2 by Friedel-Crafts acylation utilizingm-tolyl chloride in 26% yield, and subsequently brominated to give 4d in93% yield. The condensation of substituted 2-aminophenols with 4a,d inethanol gave pyrroles 5d-h in 96% (R¹═CH₃, R²═R³═H), 96% (R¹═R²═R³═H),88% (R¹═H, R²═Cl, R³═NO₂), 94% (R¹═R³═H, R²═NO₂) and 98% (R¹═R³=H,R²═COOEt) yields, respectively. Pyrroles 5d-h were then condensed withsubstituted furfurals using catalytic acid at 150° C. to giveBPO-(16-22, 24-26) in yields of 43-84%. BPO-25 was saponified utilizingKOH in THF and water to give BPO-27 in 83% yield after acid work-up.

In an experiment intended to improve yield, bromination of ketone 3a wasconducted under strict anhydrous conditions. Ketone 3a was refluxed inCH₂Cl₂ and bromine (1 eq), but after several hours TLC showed littleproduct. Serendipitously, when several drops of wet CH₂Cl₂ were added tothe reaction, a remarkably rapid discharge of the bromine color andevolution of fuming HBr gas occurred. After several minutes TLCindicated a near quantitative yield of 4a, which spontaneouslycrystallized when dried in vacuo. When 4a was condensed withN-methyl-1,2-phenylenediamine, the more reactive secondary amine canundergo alkylation by displacement of the readily accessible bromide of4a. The high regioselectivity evident in the 83% isolated yield of 5csuggests that the formation of the pyrrole ring in the initial reactionis one of imine formation rather than alkylation, despite the crowdedreaction center.

The analogs were efficiently synthesized in 5-6 steps with 11-61%overall yield.

Example 2 Synthesis of BPO-27

Modification of PPQ-102 by bromine substitution at the 5-position of thefuran ring, replacement of the secondary amine with an ether bridge, andcarboxylation, gave BPO-27. Synthesis of BPO-27 is described below.

1,3,6-Trimethyl-1H,3H-pyrimidine-2,4-dione (2) See, e.g., Azas et al.,Farmaco. 58:1263-70 (2003). In a 250 mL round bottom flask,6-methyluracil (1; 15.0 g, 119 mmol) and NaOH (9.55 g, 239 mmol) weredissolved in water (150 mL) at low heat. The solution was brought to 25°C. in an ice bath and maintained at 25° C. while dimethyl sulfate (23mL, 30.59 g, 243 mmol) was added dropwise over 20 min with vigorousstirring. After 22 h, the reaction mixture contained a white precipitateand had pH 9. NaOH (5.0 g, 125 mmol) was added to make the solutionhomogenous, and an ice bath was used to maintain a temperature of 25° C.while dimethyl sulfate (12 ml, 15.96 g, 127 mmol) was added dropwiseover 10 min. The reaction was stirred for 72 h, then NaOH (2 g, 50 mmol)was added and the reaction extracted with CHCl₃ (5×50 mL). Thechloroform extracts were pooled, dried over Na₂SO₄, and concentrated invacuo to yield 2 (18 g, 98% yield); mp 114-115° C. ¹H NMR (600 MHz,CDCl₃) δ 5.67 (s, 1H), 3.45 (s, 3H), 3.35 (s, 3H), 2.29 (s, 3H). ¹³C NMR(151 MHz, CDCl₃) δ 162.62, 152.53, 151.81, 101.16, 31.89, 28.11, 20.58.MS (ES+) (m/z): [M+1]⁺ calculated for C₇H₁₁N₂O₂, 155.17. found 155.14.

5-Benzoyl-1,3,6-trimethylpyrimidine-2,4(1H,3H)-dione (3a) See, e.g.,Tsupak et al., Chem. Heterocycl. Comp. 39:953-959 (2003). In a 100 mLround bottom flask equipped with a condenser and an air lock was added 2(5.00 g, 32.4 mmol); anhydrous zinc chloride (see, e.g., Pray inInorganic Syntheses, J. Wiley & Sons: New York, 1990; Vol. XXVIII, pp321-22) (freshly dried, 4.45 g, 32.6 mmol); dry chlorobenzene (20 mL);and benzoyl chloride (freshly distilled, 4 ml, 4.84 g, 34.4 mmol). Thereaction was refluxed in an oil bath equipped with an air lock andvigorously stirred for 3 h. The reaction was allowed to cool and thecondenser arranged for distillation. Water (40 mL) was added dropwise atfirst and then with increasing speed. Chlorobenzene (30 mL) and waterwere distilled off and the mixture was cooled on ice. Diethyl ether (30ml) was added while stirring causing a precipitate to form. Theprecipitate was collected by filtration and recrystallized from2-propanol (50 mL) to yield 3a (5.53 g, 66%); mp 143.2-143.7° C. ¹H NMR(600 MHz, CD₃CN) δ 8.74 (d, J=7.1, 2H), 8.46 (t, J=6.0, 1H), 8.37-8.29(m, 2H), 4.25 (s, 3H), 4.07 (s, 3H), 2.99 (s, 3H). ¹³C NMR (151 MHz,CD₃CN) δ 195.15, 162.17, 153.72, 153.16, 139.21, 134.93, 130.49, 130.04,113.43, 32.82, 28.61, 18.26. MS (ES+) (m/z): [M+1]⁺ calculated forC₁₄H₁₅N₂O₃, 259.28. found 259.11.

5-Benzoyl-6-(bromomethyl)-1,3-dimethylpyrimidine-2,4(1H,3H)-dione (4a).

See, e.g., Tsupak et al., supra. In a 50 mL round bottom flask 3a (1.00g, 38.7 mmol) was dissolved in CCl₄(5 mL) and CH₂Cl₂ (4 mL) at 50° C.The starting material was poorly soluble in CCl₄, and CH₂Cl₂ can besubstituted entirely. The reaction under anhydrous conditions was slowalthough the addition of a few drops of wet solvent resulted inquantitative yield in a few minutes. Bromine (200 μL, 0.621 g, 38.8mmol) was mixed with CCl₄ (5 mL) and CH₂Cl₂ (5 mL) in an addition funneland added dropwise to the solution of 3a such that the brown colordisappeared between drops. The last few drops caused the reaction toremain brown. The reaction was then brought to reflux for 10 min beforethe brown color was discharged by the addition of a few drops ofacetone. The reaction was refluxed for 30 min to remove HBr. Thereaction was quantitative by TLC and the product crystallized whenconcentrated in vacuo to yield 4a (1.305 g, 100%). The product wasrecrystallized from 2-propanol as colorless needles; mp 171.0-171.7° C.¹H NMR (600 MHz, CD₂Cl₂) δ 7.88-7.80 (m, 2H), 7.65-7.60 (m, 1H),7.52-7.45 (m, 2H), 4.24 (s, 2H), 3.59 (s, 3H), 3.31 (s, 3H). ¹³C NMR(151 MHz, CD₂Cl₂) δ 192.89, 160.94, 152.01, 149.58, 137.79, 134.46,129.78, 129.15, 114.75, 31.96, 28.59, 23.67. MS (ES+) (m/z): [M+1]⁺calculated for C₁₄H₁₄BrN₂O₃, 338.18. found 338.81.

Ethyl 3-amino-4-hydroxybezoate. In a 300 mL round bottom flask wasplaced, 3-amino-4-hydroxybezoic acid (5.50 g, 35.9 mmol), ethanol (200mL), and a magnetic stir bar. The mixture was cooled in an ice bath to10° C. and anhydrous HCl gas was bubbled through the stirred mixtureuntil homogenous. The round bottom flask was then equipped with acondenser and the solution refluxed. After 1 hour the condensor wasrearranged for distillation and 125 mL of ethanol was distilled off. Theresulting solution was then dried in vacuo to give crude ethyl3-amino-4-hydroxybezoate hydrochloride (7.73 g 35.5 mmol). Water (125mL) containing NaHCO₃ (3.0 g 35.7 mmol) was slowly added to the crudeHCl salt and the resulting mixture was extracted 3×100 mL EtOAc. TheEtOAc extracts were pooled, washed with brine, and then dried overNaSO₄. The EtOAc was removed in vacuo to give ethyl3-amino-4-hydroxybezoate (5.08 g, 78.0%) as a viscous oil whichcrystallized on standing. ¹H NMR (600 MHz, CD₂Cl₂) δ 7.42 (d, J=2.0,1H), 7.37 (dd, J=2.0, 8.2, 1H), 6.76 (d, J=8.2, 1H), 5.56-5.37 (s, 1H),4.28 (q, J=7.1, 2H), 3.76 (s, 2H), 1.34 (t, J=7.1, 3H). ¹³C NMR (151MHz, CD₂Cl₂) δ 167.79, 149.37, 134.83, 123.28, 122.37, 117.91, 114.98,61.48, 14.63. MS (ES+) (m/z): [M+1]⁺ calculated for C₉H₁₂NO₃, 182.08.found 182.09.

Ethyl3-(1,3-dimethyl-2,4-dioxo-5-phenyl-3,4-dihydro-1H-pyrrolo[3,4-d]pyrimidin-6(2H)-yl)-4-hydroxybenzoate(5h). In a 100 mL round bottom flask ethyl 3-amino-4-hydroxybezoate(1.10 g, 6.07 mmol) and 4a (1.00 g, 2.98 mmol) were refluxed in ethanol(50 mL). After 2 h, the condenser was rearranged for distillation andethanol (25 mL) was distilled off. The resulting solution was slowlypoured into a vigorously stirred solution of ice cold water (200 mL) andcitric acid (50 mg) giving a pink solid precipitate. The mixture wasstirred for 10 min and then the solid was collected by filtration andrinsed with cold water to give 5h (1.23 g, 98.5%) after drying. Theproduct was recrystallized from ethanol to give pale pink needles; m.p.129.2-130° C. ¹H NMR (600 MHz, CD₂Cl₂) δ 8.58 (s, 1H), 7.89 (dd, J=2.1,8.7, 1H), 7.67 (d, J=2.1, 1H), 7.36-7.29 (m, 2H), 7.28-7.18 (m, 3H),6.97 (d, J=8.7, 1H), 4.25 (q, J=7.1, 2H), 3.27 (s, 3H), 3.17 (s, 3H),1.30 (t, J=7.1, 3H). ¹³C NMR (151 MHz, CD₂Cl₂) δ 165.40, 159.72, 156.82,151.91, 136.06, 132.09, 130.86, 130.65, 129.49, 128.99, 128.83, 127.84,125.70, 122.92, 117.65, 104.70, 103.11, 61.23, 32.02, 28.08, 14.24. MS(ES+) (m/z): [M+1]⁺ calculated for C₂₃H₂₂N₃O₅, 420.16. found 420.13.

Ethyl6-(5-Bromofuran-2-yl)-7,9-dimethyl-8,10-dioxo-11-phenyl-7,8,9,10-tetrahydro-6H-benzo[b]pyrimido[4′,5′:3,4]pyrrolo[1,2-d][1,4]oxazine-2-carboxylate(BPO-25). Pyrrole 5h (500 mg, 1.19 mmol), 5-bromofurfural (240 mg, 1.37mmol), chloroform (7 mL), TFA (10 μL, 14.8 mg, 130 μmol), and 3 Åmolecular sieves (2.0 g, 8-12 mesh beads), and a stir bar were sealed inan Emrys 10-20 mL process vial and submerged to the level of solvent inan oil bath at 150° C. (The formation of the oxazine ring produces waterthat collects as beads at or near the top of the tube during thereaction. If the reaction were scaled up such that the beads becamelarge enough to drip back into the reaction mixture, the yield wasdiminished. The addition of molecular sieves reduced this problem forreactions containing less than 500 mg of the starting pyrrole.) Thereaction was stirred for 24 min then removed from the oil bath. Once theinternal pressure had dropped the reaction vial was rapidly cooled inwater. After cooling, the reaction was filtered through celite into a 50mL recovery flask and the dried in vacuo. The residue was dissolved in aminimum amount of CH₂Cl₂ and quickly diluted with warm ethanol (25 mL).Fine crystals began to form immediately. The mixture was then placed ona rotary evaporator and the CH₂Cl₂ was removed to increase the quantityof crystals. The mixture was then chilled, filtered, and the crystalsrinsed with cold ethanol to give BPO-25 (0.500 g, 76.4%) as fine whiteneedle like crystals. No m.p. (slow decomposition). ¹H NMR (600 MHz,CD₂Cl₂) δ 7.81 (d, J=7.7, 1H), 7.68 (dd, J=1.9, 8.4, 1H), 7.63 (t,J=7.5, 1H), 7.52 (t, J=7.5, 1H), 7.34 (t, J=7.5, 1H), 7.23 (d, J=1.8,1H), 7.09 (d, J=8.4, 1H), 7.06 (d, J=7.7, 1H), 6.86 (s, 1H), 6.14 (d,J=3.4, 1H), 5.98 (d, J=2.9, 1H), 4.11 (dq, J=7.2, 10.7, 1H), 4.00 (dq,J=7.1, 10.7, 1H), 3.48 (s, 3H), 3.26 (s, 3H), 1.14 (t, J=7.1, 3H). ¹³CNMR (151 MHz, CD₂Cl₂) δ 165.03, 159.26, 151.87, 151.79, 149.08, 131.78,131.27, 130.00, 129.80, 129.68, 128.84, 128.81, 128.29, 125.40, 124.69,124.47, 124.42, 121.57, 119.64, 114.91, 112.43, 105.93, 105.67, 68.40,61.28, 32.38, 27.95, 14.20. MS (ES+) (m/z): [M+1]⁺ calculated forC₂₈H₂₃BrN₃O₆, 576.08. found 576.04.

An Emrys process vial is a commercially available thick walled vesselmuch like a test tube typically used for microwave reactions that may besealed using a disposable plastic-lined metal cap. Precautions should betaken because the reaction vessel is under pressure when heated.

6-(5-Bromofuran-2-yl)-7,9-dimethyl-8,10-dioxo-11-phenyl-7,8,9,10-tetrahydro-6H-benzo[b]pyrimido[4′,5′:3,4]pyrrolo[1,2-d][1,4]oxazine-2-carboxylicacid (BPO-27). In a 500 mL round bottom flask equipped with stir bar,BPO-25 (1.00 g, 1.73 mmol) was dissolved in THF (100 mL) with gentleheat and then allowed to cool. A mixture of water (70 mL) and KOH (768mg, 13.7 mmol) was quickly added to the vigorously stirred solutionwhich formed a white suspension. After 3 days the mixture was ahomogenous yellow solution free of BPO-25 as determined by LC/MS. TheTHF was removed using a rotary evaporator and warm water bath leavingbehind a viscous aqueous solution. The solution was made strongly acidicto litmus using 1% aq. HCl while stirring vigorously with a glass rod.The resulting gel was shaken with EtOAc (125 mL) and then quickly pouredinto a 1 liter separatory funnel where a precipitate formed in theorganic layer. The 500 mL flask was rinsed with additional EtOAc (125mL), and this EtOAc rinse was also added to the separatory funnel.Additional EtOAc (400 mL) was added to the funnel, and the mixture wasvigorously shaken until all solids dissolved. After settling, the loweryellow aqueous layer was discarded. The EtOAc layer was washed withbrine, dried over Na₂SO₄ and dried on a rotary evaporator. The resultingslightly yellow amorphous powder was loosened by swirling with CH₂Cl₂(15 mL) and then diluted with diethyl ether (15 mL). The solids werecollected by filtration and rinsed with CH₂Cl₂:Et₂O (1:1′) to giveBPO-27 (791 mg, 83.2%) as a white solid. No m.p. (slow decomposition).¹H NMR (600 MHz, 91% CD₂Cl₂, 9% DMSO-d₆) δ 12.30 (s, 1H), 7.79 (d,J=7.7, 1H), 7.63 (dd, J=1.9, 8.4, 1H), 7.58 (t, J=7.6, 1H), 7.46 (t,J=7.5, 1H), 7.29 (t, J=7.5, 1H), 7.17 (d, J=1.7, 1H), 7.04 (d, J=8.4,1H), 7.01 (d, J=7.7, 1H), 6.89 (s, 1H), 6.12 (d, J=3.4, 1H), 5.96 (d,J=3.3, 1H), 3.44 (s, 3H), 3.21 (s, 3H). ¹³C NMR (151 MHz, 91% CD₂Cl₂, 9%DMSO-d₆) δ 166.69, 159.13, 151.86, 151.62, 148.96, 131.75, 131.00,129.74, 129.65, 129.45, 128.59, 128.53, 128.45, 125.33, 125.23, 124.38,124.20, 121.86, 119.42, 114.84, 112.34, 105.95, 105.66, 68.20, 32.28,27.84. MS (ES+) (m/z): [M+1]⁺ calculated for C₂₆H₁₉BrN₃O₆, 548.05. found548.00.

Example 2A Separation of Enantiomers of BPO-27

Separation of Enantiomers—Separation of approximately 1.0 gram ofracemic BPO-27 was carried out by Averica Discovery Services Inc.Preparative scale chiral SFC was carried out on a RegisCell 3.0×25.0 cmcolumn using an isocratic method: 75% CO₂, and 25% ethanol containing 1%isopropylamine, 80 mL/min, 100 bar, 25° C. Analysis of the separatedenantiomers was carried out on a RegisCell 4.6×100 mm column, using anisocratic method: 75% CO₂, 25% ethanol with 0.1% isopropylamine, 4mL/min, 100 bar, 25° C. Two distinct peaks were seen followingchromatography (FIG. 4A). Fraction 1 contained 413 mg with 99.5% e.e.(FIG. 4B), and fraction 2 contained 396 mg with 98.6% e.e. (FIG. 4C).

Crystal Preparation—Fraction 1, BPO-27 isopropylamine carboxylate (30mg, 49 mmol) was placed in a 75 ml separatory funnel filled with verydilute HCl (aq) (20 ml) and EtOAc (20 ml). The mixture was shakenvigorously until no undissolved solids remained. The EtOAc layer wascollected and extracted with brine and then dried over Na₂SO₄. The EtOAcwas evaporated in a 50 ml recovery flask on a rotory evaporator and thendried under high vacuum to give a white solid (24 mg, 44 mmol, yield89%). To the recovery flask was added a magnetic stir bar, DMAP (1 mg, 8mmol), EDAC (9 mg, 47 mmol) and finally dry DCM (15 ml). Once thestirred solution became homogenous, dry ethanol (10 μL, 7.9 mg, 172mmol) was added and the reaction was stirred for 24 h sealed with arubber septum. The reaction mixture was then transferred to a separatoryfunnel and extracted with dilute citric acid (10 ml). The organic layerwas collected in a 50 ml beaker, dried over Na₂SO₄ and then evaporatedin a 50 ml recovery flask on a rotary evaporator. The residue, whichshared the same R_(f) value as racemic BPO-25 (BPO-27 ethyl ester) onTLC, was purified by flash chromatography (EtOAc:hexanes, 2:3) to give aclear residue (20 mg, 35 mmol, yield 79%). The residue was dissolved in0.5 ml toluene and placed in a 1 dram vial. The vial was placed uprightin a jar filled with 3-4 mm of mixed hexanes and then sealed. The setupwas placed for 6 d in a quiet, vibration-free room, yieldingdiffraction-quality crystals, which by x-ray analysis, revealed thecompound to be (S)-BPO-27 ethyl ester [(S)-BPO-25]. See FIG. 5.

X-Ray Crystallography—Optically centered on a Bruker Duo APEXII CCDsystem a colorless crystalline needle of fraction 2 BPO-27 ethyl esterwith approximate orthogonal dimensions 0.31×0.04×0.04 mm³ was cooled to−183° C. (90° K). Indexing of the unit cell used a random set ofreflections collected from three series of 0.5° wide co-scans, 10 s perframe, and 30 frames per series that were well distributed in reciprocalspace. Data were collected [CuKα] with 0.5° wide scans, variable timeper frame dependent upon detector 2θ angle and varying ϕ and omegaangles such that nearly all unique reflections were collected at leastonce. The crystal to detector distance was 4.96 cm, thus providing acomplete sphere of data to 2θ_(max)136.52. Crystallographic calculationswere performed on an iMac with 2.80 GHz quad core processor and 8 GB ofextended memory. A total of 18750 reflections were collected andcorrected for Lorentz and polarization effects with SAINT and absorptionusing crystal faces and Blessing's method as incorporated into theprogram SADABS (Blessings, R. H. An empirical Correction for AbsorptionAnisotropy. Acta Cryst, 1995, A51, 33-38; Sheldrick, G. M. (2002)SHELXTL. Version 2008/1, ‘Siemens Area Detector Absotion Correction’Universitat Gottingen: Gottingen, Germany) with 4943 unique for pointgroup 222. The SHELXTL program package was implemented to determine theprobable space group and set up the initial files. System symmetry,systematic absences, and intensity statistics indicated thenon-centrosymmetric orthorhombic space group P2₁2₁2₁ (no. 19). Thestructure was determined by direct methods with the successful locationof a majority of the main molecule using the program XS (Sheldrick, G.M. (1997) SHELXS97 and SHELXL97. Universität Göttingen: Göttingen,Germany). The structure was refined with XL (Sheldrick, G. M. (1997)SHELXS97 and SHELXL97. Universitat Göttingen: Göttingen, Germany). Thedata collected were merged for least squares refinement to 4752 uniquedata [R(int)=0.0925]. A series of least-squares difference-Fouriercycles were required to locate the remaining non-hydrogen atoms andoptimize the full occupancy, disordered solvent toluene molecule. Allnon-hydrogen atoms were refined anisotropically. Hydrogen atoms wereidealized throughout the final refinement stages. The final structurewas refined to convergence with R(F)=9.09%, wR(F2)=22.64%, GOF=1.128 forall 4752 unique reflections [R(F)=8.86, wR(F2)=22.46% for those 4529data with Fo >4σ(Fo)]. The final difference-Fourier map was featurelessindicating that the structure is both correct and complete. An empiricalcorrection for extinction was also attempted and found to be negativeand therefore not applied. The absolute structure parameters, Flack(x)(Flack, Acta Cryst. 1983, A39, 876-881), was refined and found to be−0.08(4) while the Hooft (Hooft, R. W. W, Strayer, L. H. & Spek, A. L.J. Appl, Cryst. (2008), 41, 96-103) parameter is −0.085(18) indicatingthat the structure's absolute configuration has been reliablydetermined. Table C shows the Crystal data and structure refinement forBPO-25 [C₂₈H₂₂N₃O₆Br][C₇H₈]_(0.5).

TABLE C Identification code jfZ111of Empirical formula C31.50 H26 Br N3O6 Formula weight 622.46 Temperature 90(2) K Wavelength 1.54178 ÅCrystal system Orthorhombic Space group P 2₁2₁2₁ Unit cell dimensions a= 5.7866(4) Å α = 90°. b = 20.7586(14) Å β = 90°. c = 22.5740(14) Å γ =90°. Volume 2711.6(3) Å³ Z 4 Density (calculated) 1.525 Mg/m³ Absorptioncoefficient 2.507 mm⁻¹ F(000) 1276 Crystal size 0.31 × 0.04 × 0.04 mm³Crystal color and habit Colorless Needle Diffractometer Bruker APEX-IICCD Theta range for data collection 2.89 to 66.59°. Index ranges −6 <= h<= 6, −24 <= k <= 24, −26 <= l <= 26 Reflections collected 18348Independent reflections 4752 [R(int) = 0.0925] Observed reflections (I >2sigma(I)) 4529 Completeness to theta = 66.59° 99.7% Absorptioncorrection Numerical Max. and min. transmission 0.9130 and 0.5123Solution method SHELXS-97 (Sheldrick, 2008) Refinement method SHELXL-97(Sheldrick, 2008) Data/restraints/parameters 4752/2/420 Goodness-of-fiton F² 1.128 Final R indices [I > 2sigma(I)] R1 = 0.0886, wR2 = 0.2246 Rindices (all data) R1 = 0.0909, wR2 = 0.2264 Absolute structureparameters; −0.08(4), −0.085(18) Flack, Hooft. Largest diff. peak andhole 1.730 and −0.810 e.Å⁻³

Example 3 Synthesis of PPQ-102

Synthesis of 2, 3a, and 4a was performed as described in Example 2.Synthesis of intermediate 5a was performed as follows.

6-(2-Aminophenyl)-1,3-dimethyl-5-phenyl-1H-pyrrolo[3,4-d]pyrimidine-2,4(3H,6H)-dione(5a). 1,2-Phenylenediamine (1.320 g, 12.2 mmol) in absolute EtOH (25 mL)was stirred in a 50 mL round bottom flask. The mixture was warmed on anoil bath until homogenous, and then 4a (2.055 g, 6.1 mmol) was addedwith the aid of a powder funnel and EtOH (5 mL). The solution wasstirred vigorously at refluxed for 1 h, during which a white precipitateformed. The reaction was then cooled in an ice bath, filtered, and theprecipitate washed with cold ethanol to yield 5a (2.0357 g, 96.5%) as awhite powder. ¹H NMR (600 MHz, DMSO-d6) δ 7.37-7.31 (m, 2H), 7.25-7.19(m, 3H), 7.08-7.02 (m, 1H), 6.92 (s, 1H), 6.83 (dd, J=1.3, 7.8, 1H),6.75 (d, J=8.1, 1H), 6.48-6.43 (m, 1H), 5.08 (s, 2H), 3.33 (s, 3H), 3.19(s, 3H). ¹³C NMR (151 MHz, DMSO-d₆) δ 159.07, 150.89, 144.76, 133.51,130.42, 129.49, 129.36, 129.05, 128.64, 127.89, 127.13, 123.10, 115.65,115.46, 105.49, 102.23, 31.50, 27.40. MS (ES+) (m/z): [M+1]⁺ calculatedfor C₂₀H₁₉N₄O₂, 347.39. found 347.05.

7,9-Dimethyl-6-(5-methylfuran-2-yl)-11-phenyl-5,6-dihydropyrimido[4′,5′-3,4]pyrrolo[1,2-a]quinoxaline-8,10-(7H,9H)-dione(PPQ-102)

In a 25 mL pear shaped flask was placed 5a (1.00 g, 2.89 mmol),5-methylfurfural (332 mg, 0.30 mL, 3.02 mmol), a small crystal ofp-toluene-sulfonic acid and 1,2-dichloroethane (10 mL). The mixture wasstirred at reflux for 5 min until homogenous, and then concentrated invacuo to yield a brown sold. The solid was then recrystallized fromethanol to yield PPQ-102 (1.05 g, 83%) as slightly yellow crystals. Theproduct was recrystallized to give white crystals; m.p. 245-246° C. ¹HNMR (600 MHz, CD₂Cl₂) δ 7.81 (d, J=7.6, 1H), 7.56 (t, J=7.5, 1H), 7.45(t, J=7.5, 1H), 7.29 (t, J=7.5, 1H), 7.02 (d, J=7.6, 1H), 6.95-6.90 (m,1H), 6.83 (dd, J=1.1, 7.9, 1H), 6.48 (d, J=7.9, 1H), 6.45-6.40 (m, 1H),6.01 (s, 1H), 5.75 (d, J=2.1, 1H), 5.70 (d, J=3.0, 1H), 4.93 (d, J=2.0,1H), 3.55 (s, 3H), 3.27 (s, 3H), 2.20 (s, 3H). ¹³C NMR (151 MHz, CD₂Cl₂)δ 159.59, 152.98, 152.03, 151.67, 137.36, 132.01, 130.96, 130.17,130.06, 129.08, 128.49, 128.27, 126.49, 124.82, 123.35, 121.10, 119.45,117.42, 110.32, 109.74, 106.21, 105.24, 48.76, 32.04, 27.90, 13.70. MS(ES+) (m/z): [M+1]⁺ calculated for C₂₆H₂₃N₄O₃, 439.18. found 439.12.

Example 4 Synthesis of PPQ-1

7,9-Dimethyl-6-(5-methylfuran-2-yl)-5-(methylsulfonyl)-11-phenyl-5,6-dihydropyrimido[4′,5′-3,4]pyrrolo[1,2-a]quinoxaline-8,10-(7H,9H)-dione(PPQ-1).

Synthesis of PPQ-102 was performed as described in Example 3. In a 5 mLpear shaped flask PPQ-102 (21 mg, 48.9 μmol) was dissolved in drychloroform (1 mL). Methanesulfonyl chloride (5.5 μL, 7.7 mg, 67.2 μmol)was added followed by triethylamine (14 μL, 10.2 mg, 100 μmol), and thereaction was stirred for 30 min. TLC showed starting material soadditional methanesulfonyl chloride (5.5 μL, 7.7 mg, 67.2 μmol) andtriethylamine (14 μL, 10.2 mg, 100 μmol) were added. After 1 h TLCshowed minimal starting material. The reaction was quenched and thenpurified by TLC-prep to give PPQ-1 (17.9 mg, 72.5%) as an amorphoussolid. ¹H NMR (600 MHz, CD₂Cl₂) δ 7.84 (d, J=7.6, 1H), 7.60 (t, J=7.3,1H), 7.52-7.46 (m, 2H), 7.28 (t, J=7.3, 1H), 7.17-7.12 (m, 1H),6.98-6.93 (m, 1H), 6.87-6.79 (m, 2H), 6.62 (d, J=8.3, 1H), 5.74 (d,J=3.1, 1H), 5.72 (s, 1H), 3.56 (s, 3H), 3.27 (s, 3H), 2.63 (s, 3H), 2.20(s, 3H). ¹³C NMR (151 MHz, CD₂Cl₂) δ 159.36, 154.22, 151.76, 147.04,132.08, 131.48, 130.59, 129.87, 129.78, 129.70, 129.63, 128.85, 128.40,127.85, 127.73, 126.98, 124.94, 121.86, 111.93, 108.78, 106.40, 105.74,52.08, 37.83, 32.39, 28.00, 13.77. MS (ES+) (m/z): [M+1]⁺ calculated forC₂₇H₂₅N₄O₅S, 517.15. found 517.10.

Example 5 Synthesis of PPQ-2

5-(2-Chloroacetyl)-7,9-dimethyl-6-(5-methylfuran-2-yl)-11-phenyl-5,6-dihydropyrimido[4′,5′-3,4]pyrrolo[1,2-a]quinoxaline-8,10-(7H,9H)-dione(PPQ-2).

In a 15 mL pear shaped flask PPQ-102 (163 mg, 371 μmol) (see Example 3for synthesis of PPQ-102) was dissolved in dry CH₂Cl₂ (3 mL).Triethylamine (80 μL, 58 mg, 574 μmol) was added followed bychloroacetyl chloride (30 μL, 43 mg, 377 μmol) and the reaction wasstirred for 1 h. The reaction was found to be incomplete by TLC soadditional chloroacetyl chloride (10 μL, 14.2 mg, 126 μmol) was added.After 1 h, TLC indicated no remaining starting material. The reactionwas extracted with dilute citric acid, dried over NaSO₄, and dilutedwith ethanol (9 mL). The solution was placed on a rotary evaporatoruntil a beige precipitate ceased to form. The mixture was then cooled inan ice bath and the precipitate was collected by filtration, washed withethanol, and dried in vacuo to give PPQ-2 (170 mg, 89%) ¹H NMR (600 MHz,CD₂Cl₂) δ 7.86 (d, J=7.1, 1H), 7.58 (t, J=7.1, 1H), 7.55 (s, 1H), 7.47(t, J=7.5, 1H), 7.33-7.25 (m, J=1.2, 7.9, 2H), 7.14 (td, J=1.2, 7.8,1H), 7.02 (d, J=7.1, 1H), 6.97-6.92 (m, 1H), 6.64 (dd, J=1.0, 8.3, 1H),5.71-5.66 (m, 2H), 4.41 (dd, J=13.3, 62.4, 2H), 3.58 (s, 3H), 3.27 (s,3H), 2.16 (s, 3H). ¹³C NMR (151 MHz, CD₂Cl₂) δ 165.09, 159.44, 153.82,151.78, 147.81, 132.01, 131.13, 130.15, 129.80, 129.45, 129.01, 128.62,128.39, 127.39, 126.73, 126.01, 124.66, 122.57, 111.58, 110.34, 106.22,105.44, 47.66, 42.19, 32.24, 27.97, 13.7.1. MS (ES+) (m/z): [M+1]⁺calculated for C₂₈H₂₄ClN₄O₄, 515.15. found 515.07.

Example 6 Synthesis of PPQ-3

5-Acetyl-7,9-dimethyl-6-(5-methylfuran-2-yl)-11-phenyl-5,6.-dihydropyrimido[4′,5′-3,4]pyrrolo[1,2-a]quinoxaline-8,10-(7H,9H)-dione(PPQ-3).

In a 5 mL round bottom flask PPQ-102 (50 mg, 114 μmol) (see Example 3for synthesis of PPQ-102) and DMAP (1.2 mg, 9.8 μmol) were stirredvigorously in acetic anhydride (2.16 g, 2.0 mL, 2.12 mmol) at 100° C.for 24 h. The reaction was then poured into water (20 mL) whilestirring, alkalinized with sodium carbonate, and stirred at roomtemperature for 30 min. The mixture was then extracted with CH₂Cl₂ (3×15mL), the organic layers combined and dried over Na₂SO₄. The solvent wasremoved on a rotary evaporator and the residue purified by TLC prep togive PPQ-3 (49.0 mg, 89.4%) as a white solid after drying under highvacuum; m.p. 245-246° C. ¹H NMR (600 MHz, CD₂Cl₂) δ 7.85 (d, J=7.6, 1H),7.62 (s, 1H), 7.57 (t, J=7.5, 1H), 7.46 (t, J=7.5, 1H), 7.29 (t, J=7.5,1H), 7.25 (d, J=7.9, 1H), 7.12 (t, J=7.7, 1H), 7.02 (d, J=7.6, 1H), 6.88(t, J=7.9, 1H), 6.61 (d, J=8.3, 1H), 5.68 (d, J=2.8, 1H), 5.66 (d,J=3.0, 1H), 3.59 (s, 3H), 3.26 (s, 3H), 2.32 (s, 3H), 2.16 (s, 3H). ¹³CNMR (151 MHz, CD₂Cl₂) δ 168.72, 159.44, 153.42, 151.71, 148.56, 131.96,130.53, 130.22, 130.08, 129.88, 129.48, 129.24, 128.51, 128.22, 126.79,126.25, 126.22, 124.17, 122.13, 110.99, 110.93, 106.01, 105.15, 46.12,32.13, 27.89, 22.59, 13.68. MS (ES+) (m/z): [M+1]⁺ calculated forC₂₈H₂₅N₄O₄, 481.19. found 481.08.

Example 7 Synthesis of PPQ-4

7,9-Dimethyl-6-(5-methylfuran-2-yl)-5-nitroso-11-phenyl-5,6.-dihydropyrimido[4′,5′-3,4]pyrrolo[1,2-a]quinoxaline-8,10-(7H,9H)-dione(PPQ-4).

In a 5 mL pear shaped flask PPQ-102 (25 mg, 57 μmol) (see Example 3 forsynthesis of PPQ-102) was dissolved in dichloromethane (2 mL), and 90%t-butyl nitrite (15.6 mg, 18 μL, 136 μmol) was added. The reaction wasmaintained at room temperature for 30 min, and then dried in vacuo. Theresidue was purified by TLC prep to give PPQ-4 (21 mg, 78.8%) as a foamafter drying under high vacuum. ¹H NMR (600 MHz, CD₂Cl₂) δ 7.89 (s, 1H),7.83 (d, J=7.7, 1H), 7.76 (d, J=8.0, 1H), 7.60 (t, J=7.6, 1H), 7.50 (t,J=7.5, 1H), 7.32 (t, J=7.5, 1H), 7.25 (t, J=7.7, 1H), 7.04-6.99 (m, 2H),6.72 (d, J=8.4, 1H), 5.83 (d, J=3.1, 1H), 5.74 (d, J=2.6, 1H), 3.61 (s,3H), 3.24 (s, 3H), 2.12 (s, 3H). 13C NMR (151 MHz, CD₂Cl₂) δ 159.25,154.07, 151.80, 147.00, 131.92, 131.57, 130.23, 129.99, 129.64, 129.02,128.75, 128.59, 127.65, 127.09, 126.22, 125.56, 121.70, 120.79, 111.19,106.94, 106.39, 106.02, 43.31, 32.13, 27.97, 13.68. MS (ES+) (m/z):[M+1]⁺ calculated for C₂₆H₂₂N₅O₄, 468.17. found 468.08.

Example 8 Synthesis of PPQ-5

6-(N-methyl-2-aminophenyl)-1,3-dimethyl-5-phenyl-1Hpyrrolo[3,4-d]pyrimidine-2,4(3H,6H)-dione(5c). In a 25 mL round bottom flask N-methyl-1,2-phenylenediamine (200μl, 215 mg, 1.76 mmol) and EtOH (10 mL) were stirred. Ketone 4a (296 mg,878 μmol) (see Example 2 for synthesis of 4a) was added and the mixturewas stirred vigorously at reflux for 30 min during which a precipitateformed. The reaction was then chilled in an ice bath. The precipitatewas collected by filtration and washed with cold ethanol to yield 5c(0.263 g, 83%) as an off-white powder. ¹H NMR (600 MHz, DMSO-d6) δ7.30-7.26 (m, 2H), 7.24-7.16 (m, 4H), 6.93 (d, J=1.0, 1H), 6.86 (d,J=7.7, 1H), 6.62 (d, J=8.3, 1H), 6.49 (t, J=7.5, 1H), 5.07 (d, J=4.8,1H), 3.33 (s, 3H), 3.20 (s, 3H), 2.64 (d, J=4.9, 3H). ¹³C NMR (151 MHz,DMSO-d6) δ 159.08, 150.91, 145.66, 133.75, 130.36, 129.99, 129.34,129.10, 128.58, 127.95, 127.15, 123.52, 114.95, 110.52, 105.63, 102.41,31.56, 29.65, 27.41. MS (ES+) (m/z): [M+1]⁺ calculated for C₂₁H₂₁N₄O₂,361.17. found 361.10.

5,7,9-Trimethyl-6-(5-methylfuran-2-yl)-11-phenyl-5,6-dihydropyrimido[4′,5′-3,4]pyrrolo[1,2-a]quinoxaline-8,10-(7H,9H)-dione(PPQ-5). In a 10 mL pear shaped flask was placed 5c (100 mg, 277 mmol),5-methyl-2-furaldehyde (39 μL, 43.1 mg, 392 μmol), a magnetic stir bar,a small crystal of p-toluene-sulfonic acid, and 1,2-dichloroethane (3mL). The mixture was refluxed until homogenous, with no startingmaterial remaining as determined by TLC. The reaction was thenconcentrated in vacuo and diluted with ethanol (7 mL) causing aprecipitate to form. The precipitate was collected by filtration andwashed with cold ethanol to yield PPQ-5 (98.6 mg, 78.5%), as a whitewaxy solid; m.p. 174-175° C. ¹H NMR (600 MHz, CD₂Cl₂) δ 7.83 (d, J=7.5,1H), 7.56 (t, J=7.5, 1H), 7.44 (t, J=7.5, 1H), 7.27 (t, J=7.4, 1H),7.07-7.01 (m, 1H), 6.96 (d, J=7.5, 1H), 6.76 (d, J=7.3, 1H), 6.60-6.53(m, 1H), 6.51-6.44 (m, 1H), 5.82 (s, 1H), 5.72 (d, J=2.2, 1H), 5.58 (d,J=3.1, 1H), 3.55 (s, 3H), 3.25 (s, 3H), 3.10 (s, 3H), 2.16 (s, 3H). ¹³CNMR (151 MHz, CD₂Cl₂) δ 159.63, 152.80, 151.97, 150.12, 139.23, 132.10,130.94, 130.05, 129.59, 128.97, 128.47, 128.20, 126.71, 125.50, 122.36,121.08, 118.70, 115.02, 112.17, 109.76, 106.14, 105.23, 55.89, 38.32,32.37, 27.89, 13.73. MS (ES+) (m/z): [M+1]⁺ calculated for C₂₇H₂₅N₄O₃,453.19. found 453.17.

Example 9 Synthesis of PPQ-6

7,9-Dimethyl-6-(5-ethylfuran-2-yl)-11-phenyl-5,6-dihydropyrimido[4′,5′-3,4]pyrrolo[1,2-a]quinoxaline-8,10-(7H,9H)-dione(PPQ-6).In a 5 mL pear shaped flask was placed 5a (50 mg, 144 μmol) (seeExample 3 for synthesis of 5a), 5-ethyl-2-furaldehyde (17.5 μL, 18.4 mg,149 μmol), a small crystal of p-toluene-sulfonic acid and1,2-dichloroethane (1.5 mL). The mixture was refluxed for 34 min,concentrated in vacuo, and triturated with 2-butanone to give a whitewaxy precipitate. The precipitate was collected by filtration and washedwith cold 2-butanone to yield PPQ-6 (37 mg, 57%); m.p. 224° C. ¹H NMR(600 MHz, CD₂Cl₂) δ 7.80 (d, J=7.6, 1H), 7.56 (t, J=7.5, 1H), 7.45 (t,J=7.5, 1H), 7.35 (s, OH), 7.29 (t, J=7.5, 1H), 7.02 (d, J=7.7, 1H),6.94-6.89 (m, 1H), 6.82 (dd, J=1.2, 7.9, 1H), 6.48 (d, J=7.6, 1H),6.46-6.40 (m, 1H), 6.01 (s, 1H), 5.75 (d, J=3.1, 1H), 5.72 (d, J=2.8,1H), 4.91 (d, J=2.1, 1H), 3.56 (s, 3H), 3.27 (s, 3H), 2.54 (q, J=7.6,2H), 1.12 (t, J=7.6, 3H). ¹³C NMR (151 MHz, CD₂Cl₂) δ 159.59, 158.54,152.03, 151.57, 137.43, 132.01, 130.96, 130.18, 130.06, 129.08, 128.55,128.49, 128.27, 126.50, 124.87, 123.35, 121.11, 119.47, 117.38, 110.39,109.39, 105.23, 104.50, 48.82, 32.05, 27.90, 21.51, 11.79. MS (ES+)(m/z): [M+1]⁺ calculated for C₂₇H₂₅N₄O₃, 453.19. found 453.11.

Example 10 Synthesis of PPQ-7

7,9-Dimethyl-6-(5-methylthiophene-2-yl)-11-phenyl-5,6-dihydropyrimido[4′,5′-3,4]pyrrolo[1,2-a]quinoxaline-8,10-(7H,9H)-dione(PPQ-7).

In a 5 mL pear shaped flask was placed 5a (50 mg 144 μmol) (see Example3 for synthesis of 5a), 5-methyl-2-thiophenecarbaldehyde (16 μL, 18.7mg, 148 μmol), a small crystal of p-toluene-sulfonic acid and1,2-dichloroethane (3 mL). The mixture was refluxed for 30 min,concentrated in vacuo, and diluted with ethanol (6 mL) to slowly form awaxy white solid. The precipitate was collected by filtration and washedwith cold ethanol to yield PPQ-7 (48.9 mg, 74.5%). ¹H NMR (600 MHz,CD₂Cl₂) δ 7.81 (d, J=7.6, 1H), 7.56 (t, J=7.5, 1H), 7.45 (t, J=7.5, 1H),7.29 (t, J=7.5, 1H), 7.02 (d, J=7.7, 1H), 6.95-6.90 (m, 1H), 6.78 (dd,J=1.2, 7.9, 1H), 6.53-6.42 (m, 4H), 6.21 (d, J=2.4, 1H), 4.61 (d, J=2.4,1H), 3.54 (s, 3H), 3.24 (s, 3H), 2.31 (s, 3H). ¹³C NMR (151 MHz, CD₂Cl₂)δ 159.55, 152.00, 143.36, 140.95, 136.88, 132.05, 130.94, 130.05,130.00, 129.12, 128.51, 128.29, 126.59, 125.75, 125.04, 124.96, 122.89,121.20, 119.68, 117.81, 112.59, 105.26, 50.49, 32.22, 27.89, 15.52. MS(ES+) (m/z): [M+1]⁺ calculated for C₂₆H₂₃N₄O₂S, 455.15. found 455.08.

Example 11 Synthesis of PPQ-8

7,9-Dimethyl-6-(4,5-dimethylfuran-2-yl)-11-phenyl-5,6-dihydropyrimido[4′,5′-3,4]pyrrolo[1,2-a]quinoxaline-8,10-(7H,9H)-dione(PPQ-8).

In a 5 mL pear shaped flask was placed 5a (100 mg, 289 μmol) (seeExample 3 for synthesis of 5a), 4,5-dimethyl-2-furaldehyde (37 μL, 37.6mg, 303 μmol), a small crystal of p-toluene-sulfonic acid, and1,2-dichloroethane (2 mL). The mixture was refluxed until homogenous,concentrated to dryness in vacuo and triturated with benzene and thenethanol to give PPQ-8 (89 mg, 68%) as a white waxy solid. ¹H NMR (600MHz, CD₂Cl₂) δ 7.81 (d, J=7.7, 1H), 7.56 (t, J=7.5, 1H), 7.45 (t, J=7.5,1H), 7.29 (t, J=7.5, 1H), 7.02 (d, J=7.7, 1H), 6.92 (td, J=1.3, 7.8,1H), 6.83 (dd, J=1.2, 7.9, 1H), 6.48 (d, J=8.0, 1H), 6.45-6.39 (m, 1H),5.97 (s, 1H), 5.61 (s, 1H), 4.91 (d, J=2.1, 1H), 3.54 (s, 3H), 3.27 (s,3H), 2.10 (s, 3H), 1.72 (s, 3H). ¹³C NMR (151 MHz, CD₂Cl₂) δ 159.58,152.03, 150.33, 148.13, 137.37, 131.99, 131.02, 130.10, 130.05, 129.07,128.49, 128.28, 126.46, 124.78, 123.27, 121.07, 119.37, 117.37, 114.69,112.22, 110.37, 105.25, 48.72, 31.97, 27.90, 11.48, 9.80. MS (ES+)(m/z): [M+1]⁺ calculated for C₂₇H₂₅N₄O₃, 453.19. found 453.24.

Example 12 Synthesis of PPQ-9

6(5-Chlorofuran-2-yl)-7,9-dimethyl-11-phenyl-5,6-dihydropyrimido[4′,5′-3,4]pyrrolo[1,2-a]quinoxaline-8,10-(7H,9H)-dione(PPQ-9). In a 5 mL pear shaped flask was placed 5a (100 mg, 289 μmol)(see Example 3 for synthesis of 5a), 5-chloro-2-furaldehyde (39.8 mg,305 μmol), a small crystal of p-toluene-sulfonic acid, and1,2-dichloroethane (1.5 mL). The mixture was refluxed for ˜2 min untilhomogenous, concentrated in vacuo and then diluted with ethanol (5 mL).The solution was placed in a freezer overnight to give a light orangeprecipitate. The precipitate was collected by filtration and rinsed withcold ethanol to give PPQ-9 (107.6 mg, 81.2%). No m.p. (slowdecomposition). ¹H NMR (600 MHz, CD₂Cl₂) δ 7.80 (d, J=7.6, 1H), 7.56 (t,J=7.5, 1H), 7.45 (t, 1H), 7.29 (t, J=7.5, 1H), 7.01 (d, J=7.7, 1H),6.95-6.91 (m, 1H), 6.84 (dd, J=1.1, 7.9, 1H), 6.50-6.42 (m, 2H), 6.03(s, 1H), 5.98 (d, J=3.3, 1H), 5.86 (d, J=3.3, 1H), 4.89 (s, 1H), 3.53(s, 3H), 3.26 (s, 3H). ¹³C NMR (151 MHz, CD₂Cl₂) δ 159.49, 153.07,151.99, 136.79, 136.60, 131.97, 130.80, 130.57, 130.01, 129.19, 128.52,128.32, 126.63, 124.79, 123.73, 121.12, 119.76, 117.49, 111.56, 109.12,107.16, 105.29, 48.62, 32.06, 27.92. MS (ES+) (m/z): [M+l]⁺ calculatedfor C₂₅H₂₀ClN₄O₃, 459.12. found 458.98.

Example 13 Synthesis of PPQ-10

6-(5-Bromofuran-2-yl)-7,9-dimethyl-11-phenyl-5,6-dihydropyrimido[4′,5′-3,4]pyrrolo[1,2-a]quinoxaline-8,10-(7H,9H)-dione(PPQ-10). In a 5 mL pear shaped flask was placed 5a (100 mg, 289 μmol)(see Example 3 for synthesis of 5a), 5-bromo-2-furaldehyde (53 mg, 303μmol), a small crystal of p-toluene-sulfonic acid, and1,2-dichloroethane (2 mL). The mixture was refluxed until homogenous,about 5 min, concentrated in vacuo and then diluted with ethanol (10mL). An orange precipitate slowly formed which was aided by chilling themixture in a freezer. The resulting solids were collected by filtrationand rinsed with cold ethanol to give PPQ-10 (107.2 mg, 73.7%) as anorange powder. No m.p. (slow decomposition). ¹H NMR (600 MHz, CD₂Cl₂) δ7.80 (d, J=7.6, 1H), 7.56 (t, J=7.5, 1H), 7.45 (t, J=7.5, 1H), 7.29 (t,J=7.5, 1H), 7.02 (d, J=7.7, 1H), 6.96-6.90 (m, 1H), 6.84 (dd, J=1.1,7.9, 1H), 6.48 (d, J=7.7, 1H), 6.47-6.41 (m, 1H), 6.12 (d, J=3.3, 1H),6.04 (d, J=1.7, 1H), 5.85 (dd, J=0.8, 3.3, 1H), 4.95 (d, J=2.3, 1H),3.53 (s, 3H), 3.27 (s, 3H). ¹³C NMR (151 MHz, CD₂Cl₂) δ 159.49, 155.46,151.98, 136.82, 131.97, 130.80, 130.56, 130.01, 129.18, 128.52, 128.31,126.63, 124.76, 123.70, 122.37, 121.12, 119.72, 117.48, 112.20, 111.90,109.18, 105.28, 48.62, 32.08, 27.93. MS (ES+) (m/z): [M+1]⁺ calculatedfor C₂₅H₂₀BrN₄O₃, 503.07. found 503.00.

Example 14 Synthesis of PPQ-11

7,9-Dimethyl-6-(5-iodofuran-2-yl)-11-phenyl-5,6-dihydropyrimido[4′,5′-3,4]pyrrolo[1,2-a]quinoxaline-8,10-(7H,9H)-dione(PPQ-11) In a 5 mL pear shaped flask was placed 5a (150 mg, 433 μmol)(see Example 3 for synthesis of 5a), 5-iodo-2-furaldehyde (106 mg, 478μmol), a small crystal of p-toluene-sulfonic acid, a magnetic stir bar,and 1,2-dichloroethane (2 mL). The mixture was refluxed untilhomogenous, about 5 min, concentrated in vacuo and then diluted withethanol (10 mL). A yellow precipitate slowly formed which was collectedby filtration and rinsed with cold ethanol to give PPQ-11 (223 mg,93.6%), as a light orange powder. No m.p. (slow decomposition). ¹H NMR(600 MHz, CD₂Cl₂) δ 7.80 (d, J=7.7, 1H), 7.56 (t, J=7.5, 1H), 7.45 (t,J=7.5, 1H), 7.29 (t, J=7.5, 1H), 7.02 (d, J=7.7, 1H), 6.93 (td, J=1.4,7.9, 1H), 6.83 (dd, J=1.2, 7.9, 1H), 6.48 (d, J=7.3, 1H), 6.46-6.41 (m,1H), 6.34 (d, J=3.3, 1H), 6.09 (d, J=2.0, 1H), 5.78 (d, J=3.3, 1H), 4.95(d, J=2.4, 1H), 3.53 (s, 3H), 3.26 (s, 3H). ¹³C NMR (151 MHz, CD₂Cl₂) δ159.50, 158.97, 151.98, 136.86, 131.97, 130.80, 130.52, 130.01, 129.17,128.51, 128.30, 126.62, 124.75, 123.64, 121.12, 120.97, 119.70, 117.47,112.15, 109.34, 105.28, 88.96, 48.57, 32.10, 27.93. MS (ES+) (m/z):[M+1]+ calculated for C₂₅H₂₀IN₄O₃, 551.06. found 551.01.

Example 15 Synthesis of PPQ-12

7,9-Dimethyl-11-phenyl-6-(5-trifluoromethylfuran-2-yl)-5,6-dihydropyrimido[4′,5′-3,4]pyrrolo[1,2-a]quinoxaline-8,10-(7H,9H)-dione(PPQ-12). In a 5 mL pear shaped flask was placed 5a (100 mg, 289 μmol)(see Example 3 for synthesis of 5a), 5-trifluoromethyl-2-furaldehyde (50mg, 305 μmol), a small crystal of p-toluene-sulfonic acid, and1,2-dichloroethane (2 mL). The mixture was refluxed for ˜5 min untilhomogenous, concentrated in vacuo and diluted with ethanol (8 mL). Thesolution was placed in a freezer for 4 days during which yellow crystalsformed. The crystals were filtered and rinsed with ethanol to givePPQ-12 (126 mg, 88.5%); m.p. 256-257° C. 1H NMR (600 MHz, CD2Cl2) δ 7.80(d, J=7.7, 1H), 7.56 (t, J=7.4, 1H), 7.46 (tt, J=1.2, 7.5, 1H), 7.30 (t,J=7.4, 1H), 7.02 (d, J=7.7, 1H), 6.93 (ddd, J=1.6, 7.1, 8.0, 1H), 6.85(dd, J=1.2, 7.9, 1H), 6.61 (dd, J=1.0, 3.4, 1H), 6.51-6.42 (m, J=4.1,7.1, 8.3, 2H), 6.10 (s, 1H), 5.95 (d, J=3.5, 1H), 4.96 (d, J=2.5, 1H),3.55 (s, 3H), 3.26 (s, 3H). ¹³C NMR (151 MHz, CD₂Cl₂) δ 159.47, 156.71,151.99, 141.85, 141.57, 141.29, 141.01, 136.75, 131.97, 130.76, 130.72,129.99, 129.24, 128.55, 128.34, 126.74, 124.80, 123.79, 121.71, 121.18,119.94, 119.91, 118.17, 117.43, 112.80, 112.78, 110.02, 109.09, 105.31,48.56, 32.14, 27.94. MS (ES+) (m/z): [M+1]⁺ calculated for C₂₆H₂₀F₃N₄O₃,493.15. found 493.08.

Example 16 Synthesis of PPQ-13

7,9-Dimethyl-11-phenyl-6-(5-phenylfuran-2-yl)-5,6-dihydropyrimido[4′,5′-3,4]pyrrolo[1,2-a]quinoxaline-8,10-(7H,9H)-dione(PPQ-13). In a 5 mL pear shaped flask was placed 5a (100 mg, 289 μmol)(see Example 3 for synthesis of 5a), 5-phenyl-2-furaldehyde (52 mg, 302μmol), a small crystal of p-toluene-sulfonic acid, and1,2-dichloroethane (2 mL). The mixture was refluxed for ˜5 min untilhomogenous, concentrated slightly in vacuo and diluted with ethanol (10mL). A white waxy precipitate formed almost immediately, which was thenchilled, filtered and washed with ethanol to give PPQ-13 (141.5 mg,97.9%). ¹H NMR (600 MHz, CD₂Cl₂) δ 7.81 (d, J=7.7, 1H), 7.57 (t, J=7.6,1H), 7.54-7.50 (m, 2H), 7.46 (t, J=7.5, 1H), 7.35 (t, J=7.8, 2H), 7.30(t, J=7.5, 1H), 7.24 (t, J=7.4, 1H), 7.04 (d, J=7.7, 1H), 6.92 (td,J=1.3, 7.9, 1H), 6.85 (dd, J=1.3, 7.9, 1H), 6.52 (d, J=7.5, 1H),6.48-6.44 (m, 2H), 6.14 (d, J=1.3, 1H), 5.97 (d, J=3.3, 1H), 4.94 (s,1H), 3.62 (s, 3H), 3.26 (s, 3H). 13C NMR (151 MHz, CD₂Cl₂) δ 159.55,154.06, 153.27, 152.02, 137.34, 132.03, 130.90, 130.35, 130.27, 130.04,129.13, 128.95, 128.51, 128.29, 127.88, 126.58, 124.92, 123.65, 123.48,121.20, 119.63, 117.44, 110.72, 110.15, 105.71, 105.29, 48.85, 32.22,27.91. MS (ES+) (m/z): [M+1]⁺ calculated for C₃₁H₂₅N₄O₃, 501.19. found501.13.

Example 17 Synthesis of PPQ-14

7,9-Dimethyl-11-phenyl-6-(5-hydroxymethylfuran-2-yl)-5,6-dihydropyrimido[4′,5′-3,4]pyrrolo[1,2-a]quinoxaline-8,10-(7H,9H)-dione(PPQ-14). In a 5 mL pear shaped flask was placed 5a (50 mg, 144 μmol)(see Example 3 for synthesis of 5a), 5-hydroxymethyl-2-furaldehyde (18.8mg, 149 μmol), TFA (5 μL, 7.4 mg, 65 μmol) and 1,2-dichloroethane (1.5mL). The mixture was refluxed for 10 min forming a precipitate. Thereaction was continued for 5 min before chilling in an ice bath. Theprecipitate was filtered and washed with cold 1,2-dichloroethane to givePPQ-14 (44.9 mg, 68.4%), as a white waxy solid. 1H NMR (600 MHz, CD₂Cl₂)δ 7.79 (d, J=7.5, 1H), 7.56 (t, J=7.5, 1H), 7.45 (t, J=7.5, 1H), 7.29(t, J=7.4, 1H), 7.01 (d, J=7.6, 1H), 6.93-6.90 (m, 1H), 6.83 (dd, J=1.3,7.9, 1H), 6.47 (dd, J=1.2, 8.3, 1H), 6.45-6.40 (m, 1H), 6.07-6.03 (m,2H), 5.79 (d, J=3.1, 1H), 4.90 (s, 1H), 4.47 (s, 2H), 3.54 (s, 3H), 3.25(s, 3H), 1.83 (s, 1H). ¹³C NMR (151 MHz, CD₂Cl₂) δ 159.54, 154.73,153.68, 152.02, 137.18, 131.99, 130.88, 130.36, 130.03, 129.14, 128.51,128.30, 126.56, 124.86, 123.51, 121.13, 119.62, 117.40, 109.93, 109.77,108.60, 105.27, 57.39, 48.78, 32.09, 27.90. MS (ES+) (m/z): [M+1]⁺calculated for C₂₆H₂₃N₄O₄, 455.171931. found 455.15.

Example 18 Synthesis of PPQ-15

6-(Benzofuran-2-yl)-7,9-dimethyl-11-phenyl-5,6-dihydropyrimido[4′,5′-3,4]pyrrolo[1,2-a]quinoxaline-8,10-(7H,9H)-dione(PPQ-15). In a 5 mL pear shaped flask was placed 5a (100 mg, 289 μmol)(see Example 3 for synthesis of 5a), 2-benzofurancarboxaldehyde (52 mg,302 μmol), a small crystal of p-toluene-sulfonic acid, and1,2-dichloroethane (2 mL). The mixture was refluxed for several min togive a white precipitate. The reaction was chilled in an ice bath,filtered, and the precipitate washed with cold 1,2-dichloroethane togive PPQ-15 (125 mg, 91.2%) as a white very waxy solid; m.p. 294-295° C.1H NMR (600 MHz, CD₂Cl₂) δ 7.82 (d, J=7.6, 1H), 7.57 (t, J=7.6, 1H),7.46 (t, J=7.5, 1H), 7.41 (d, J=8.3, 1H), 7.38 (d, J=7.6, 1H), 7.30 (t,J=7.5, 1H), 7.23 (t, J=7.4, 1H), 7.13 (t, J=7.4, 1H), 7.04 (d, J=7.7,1H), 6.89 (t, J=7.5, 1H), 6.85 (d, J=6.7, 1H), 6.49 (d, J=8.2, 1H), 6.42(t, J=7.1, 1H), 6.27 (s, 1H), 6.20 (s, 1H), 5.03 (s, 1H), 3.58 (s, 3H),3.27 (s, 3H). ¹³C NMR (151 MHz, CD₂Cl₂) δ 159.53, 156.19, 155.16,152.02, 136.95, 131.99, 130.88, 130.59, 130.00, 129.19, 128.55, 128.34,127.75, 126.62, 124.85, 124.82, 123.83, 123.25, 121.35, 121.13, 119.76,117.42, 111.32, 109.46, 106.07, 105.35, 49.10, 32.09, 27.93. MS (ES+)(m/z): [M+1]⁺ calculated for C₂₉H₂₃N₄O₃, 475.18. found 475.01.

Example 19 Synthesis of BPO-16

1,3-Dimethyl-6-(2-hydroxyphenyl)-5-phenyl-1H-pyrrolo[3,4-d]pyrimidine-2,4(3H,6H)-dione(5e). In a 25 mL round bottom was placed o-aminophenol (163 mg, 1.5mmol) and EtOH (10 mL). The mixture was warmed until homogenous and thencompound 4a (250 mg, 741 μmol) was added (see Example 2 for synthesis of4a). The mixture was stirred vigorously at reflux during which aprecipitate formed. After 1 h the mixture was cooled in an ice bath,filtered, and the precipitate washed with cold EtOH to yield 5e (248 mg,96.4%) as a white powder. ¹H NMR (600 MHz,) δ 10.02 (s, 1H), 7.29-7.26(m, 2H), 7.24-7.17 (m, 4H), 7.10 (dd, J=1.6, 7.8, 1H), 6.96 (s, 1H),6.90 (dd, J=1.1, 8.2, 1H), 6.77 (td, J=1.2, 7.6, 1H), 3.34 (s, 3H), 3.21(s, 3H). ¹³C NMR (151 MHz, DMSO d6) δ 159.11, 152.86, 150.91, 133.81,130.35, 129.98, 129.67, 129.13, 128.58, 127.83, 127.18, 125.95, 118.99,116.39, 106.16, 101.88, 31.50, 27.46. MS (ES+) (m/z): [M+1]⁺ calculatedfor C₂₀H₁₈N₃O₃, 348.13. found 348.11.

6-(5-Bromofuran-2-yl)-7,9-dimethyl-11-phenyl-6H-benzo[b]pyrimido[4′,5′-3,4]pyrrolo[1,2-d]oxazine-8,10-(7H,9H)-dione(BPO-16). 5-bromo-2-furaldehyde (186 mg, 1.06 mmol), 5e (300 mg, 864μmol), chloroform (6 mL) and TFA (10 μL, 14.9 mg, 130 μmol) were sealedin a Emrys 2-5 mL process vial 5 and submerged to the level of solventin an oil bath at 150° C. for 19 min. The reaction was allowed to cool,filtered through a celite plug to remove impurities, and concentrated todryness in vacuo. The residue was dissolved in a minimum volume ofCH₂Cl₂ and then diluted with ethanol (15 mL). The solution was thenplaced on a rotary evaporator to remove CH₂Cl₂. Once the solution beganto crystallize the mixture was allowed to stand. The mixture was thencooled, filtered, and the crystals rinsed with cold ethanol to giveBPO-16 (365 mg, 83.8%). No m.p. (slow decomposition). ¹H NMR (600 MHz,CD₂Cl₂) δ 7.79 (d, J=7.6, 1H), 7.58 (t, J=7.5, 1H), 7.49 (t, J=7.5, 1H),7.33 (t, J=7.5, 1H), 7.07 (d, J=7.6, 1H), 7.05-6.98 (m, 2H), 6.79 (s,1H), 6.64 (ddd, J=2.2, 6.7, 8.6, 1H), 6.53 (d, J=8.6, 1H), 6.14 (d,J=3.4, 1H), 5.98 (dd, J=0.8, 3.4, 1H), 3.47 (s, 3H), 3.26 (s, 3H). ¹³CNMR (151 MHz, CD₂Cl₂) δ 159.35, 152.32, 151.83, 145.46, 131.86, 130.86,130.19, 129.94, 129.53, 128.66, 128.49, 127.11, 125.85, 124.38, 124.11,122.54, 120.39, 119.72, 114.75, 112.35, 106.50, 105.58, 68.13, 32.42,27.94. MS (ES+) (m/z): [M+1]⁺ calculated for C₂₅H₁₉BrN₃O₄, 504.06. found504.03.

Example 20 Synthesis of BPO-17

7,9-Dimethyl-6-(5-iodofuran-2-yl)-11-phenyl-6Hbenzo[b]pyrimido[4′,5′-3,4]pyrrolo[1,2-d]oxazine-8,10-(7H,9H)-dione(BPO-17).5-iodo-2-furaldehyde (84 mg, 378 μmol), 5e (100 mg, 288 μmol)(see Example 19 for synthesis of 5e), chloroform (1 mL), and TFA (10 μL,14.9 mg, 130 μmol) were sealed in a Emrys 2-5 mL process vial andsubmerged to the level of solvent in an oil bath at 150° C. for 19 min.The reaction was cooled in an ice bath, filtered through a celite plugand dried in vacuo. The residue was then purified via flashchromatography to give BPO-17 (68.4 mg, 43%). ¹H NMR (600 MHz, CD₂Cl₂) δ7.79 (d, J=7.6, 1H), 7.58 (t, J=7.6, 1H), 7.49 (t, J=7.5, 1H), 7.33 (t,J=7.5, 1H), 7.07 (d, J=7.6, 1H), 7.05-6.98 (m, 2H), 6.84 (s, 0.7H), 6.80(s, 0.3H), 6.67-6.61 (m, J=2.3, 5.5, 6.7, 1H), 6.53 (d, J=8.0, 1H), 6.36(d, J=3.3, 0.7H), 6.15 (d, J=3.4, 0.3H), 5.99 (d, J=3.3, 0.3H), 5.92 (d,J=3.3, 0.7H), 3.47 (s, 0.74H), 3.47 (s, 2.27H), 3.26 (s, 3H). ¹³C NMR(151 MHz, CD₂Cl₂) δ 159.34, 155.77, 152.32, 151.81, 145.49, 145.45,131.85, 130.84, 130.80, 130.19, 129.94, 129.51, 128.65, 128.48, 127.09,125.84, 124.37, 124.32, 124.10, 122.53, 122.50, 121.06, 120.37, 119.70,114.89, 114.74, 112.34, 106.66, 106.49, 105.56, 91.02, 68.03, 32.42,27.93. MS (ES+) (m/z): [M+1]⁺ calculated for C₂₅H₁₉IN₃O₄, 552.04. found552.07.

Example 21 Synthesis of BPO-18

7,9-Dimethyl-6-(5-methylfuran-2-yl)-11-phenyl-6H-benzo[b]pyrimido[4′,5′-3,4]pyrrolo[1,2-d]oxazine-8,10-(7H,9H)-dione(BPO-18). 5-methylfurfural (8 μL, 8.9 mg, 80 μmol), 5e (25 mg, 72 μmol)(see Example 19 for synthesis of 5e), chloroform (3 mL) and a smallcrystal of TsOH were sealed in a Emrys 2-5 mL process vial and submergedto the level of solvent in an oil bath at 150° C. for 35 min. Once thetube cooled, the contents were purified by TLC-prep to give BPO-18 (15.6mg, 49.4%) as a waxy white powder. ¹HNMR (600 MHz, CD₂Cl₂) δ 7.80 (d,J=7.6, 1H), 7.58 (t, J=7.5, 1H), 7.48 (t, J=7.5, 1H), 7.32 (t, J=7.4,1H), 7.07 (d, J=7.6, 1H), 7.03-6.97 (m, 2H), 6.77 (s, 1H), 6.63 (ddd,J=3.1, 5.7, 8.6, 1H), 6.53 (d, J=8.0, 1H), 5.85 (d, J=3.1, 1H), 5.77 (d,J=2.2, 1H), 3.47 (s, 3H), 3.26 (s, 3H), 2.22 (s, 3H). ¹³C NMR (151 MHz,CD₂Cl₂) δ 159.44, 154.52, 151.86, 148.60, 145.78, 131.90, 130.43,130.36, 129.99, 129.42, 128.62, 128.44, 126.93, 126.01, 123.90, 122.25,120.35, 119.67, 113.05, 107.72, 106.40, 105.51, 68.58, 32.37, 27.91,13.81. MS (ES+) (m/z): [M+1]⁺ calculated for C₂₆H₂₂N₃O₄, 440.16. found440.11.

Example 22 Synthesis of BPO-19

5-m-Tolyl-1,3,6-trimethylpyrimidine-2,4(1H,3H)-dione (3d). In a 100 mLround bottom flask equipped with a condenser and an air lock was placedcompound 2 (see FIG. 3) (5.00 g, 32.4 mmol), anhydrous zinc chloride 3(4.45 g, 32.6 mmol), dry chlorobenzene (20 mL) and m-tolyl chloride (4.6mL, 5.38 g, 34.8 mmol). The reaction was brought to reflux in an oilbath and vigorously stirred for 3 h. After cooling, water (40 mL) wasadded through the condenser, dropwise at first and then with increasingspeed. The condenser was then rearranged for distillation and thechlorobenzene was removed by azeotropic distillation. The solution wasthen cooled in an ice bath, and diethyl ether (30 mL) was added whilestirring to give a precipitate. The precipitate was filtered andrecrystallized from 2-propanol to yield 3d (1.11 g, 26.3%); m.p.136.8-139° C. ¹H NMR (600 MHz, CD₂Cl₂) δ 7.68-7.66 (m, 1H), 7.65-7.62(m, 1H), 7.44-7.40 (m, 1H), 7.35 (t, J=7.6, 1H), 3.46 (s, 3H), 3.30 (s,3H), 2.40 (s, 3H), 2.18 (s, 3H). ¹³C NMR (151 MHz, CD₂Cl₂) δ 194.00,161.07, 152.22, 151.98, 139.22, 138.16, 134.94, 130.03, 129.01, 126.93,113.36, 32.36, 28.32, 21.54, 17.99. MS (ES+) (m/z): [M+1]⁺ calculatedfor C₁₅H₁₇N₂O₃, 273.12. found 237.10.

6-(Bromomethyl)-5-m-tolyl-1,3-dimethylpyrimidine-2,4(1H,3H)-dione (4d).

In a 25 mL 2-neck round bottom flask equipped with a condenser, additionfunnel and air lock, compound 3d (1.00 g, 3.67 mmol) was dissolved inCH₂Cl₂ (6 mL) and held at reflux. Bromine (200 μL, 0.624 g, 3.9 mmol)was mixed with CH₂Cl₂ (9 mL) in the addition funnel and added dropwiseto the solution of 3d at such a rate that the color was dischargedbetween drops. The last few drops caused the reaction to stay brown. Thereaction continued for 10 min before the color was discharged by theaddition of a few drops of acetone. TLC showed the reaction wasquantitative. The reaction was dried in vacuo and the remaining solidcrystallized from 2-propanol to yield 4d (1.20 g, 93%); m.p. 136.1-137°C. ¹H NMR (600 MHz, CD₂Cl₂) δ 7.67 (s, 1H), 7.63 (d, J=7.8, 1H), 7.45(d, J=7.5, 1H), 7.37 (t, J=7.6, 1H), 4.22 (s, 2H), 3.60 (d, J=6.6, 3H),3.30 (d, J=7.7, 3H), 2.41 (s, 3H). ¹³C NMR (151 MHz, CD₂Cl₂) δ 193.00,160.95, 152.09, 149.09, 139.30, 137.77, 135.37, 130.22, 129.04, 127.11,115.04, 31.98, 28.62, 23.75, 21.55. MS (ES+) (m/z): [M+1]⁺ calculatedfor C₁₅H₁₆BrN₂O₃, 351.03. found 350.99.

1,3-Dimethyl-6-(2-hydroxyphenyl)-5-m-tolyl-1H-pyrrolo[3,4-d]pyrimidine-2,4(3H,6H)-dione(5d). In a 25 mL round bottom flask 2-aminophenol (683 mg, 6.26 mmol)was dissolved in warm EtOH (10 mL) and compound 4d (296 mg, 878 μmol)was added while stirring. The reaction was refluxed 20 min to give awhite precipitate. The reaction was then cooled in an ice bath,filtered, and the precipitate washed with cold ethanol to yield 5d (1.08g, 96.3%) as a off white powder. ¹H NMR (600 MHz, DMSO-d6) δ 10.02 (s,1H), 7.20 (td, J=1.6, 8.2, 1H), 7.16 (s, 1H), 7.10-7.06 (m, 2H), 7.04(d, J=7.6, 1H), 7.01 (d, J=7.5, 1H), 6.93 (s, 1H), 6.90 (dd, J=0.8, 8.2,1H), 6.77 (t, J=7.6, 1H), 3.33 (s, 3H), 3.20 (s, 3H), 2.19 (s, 3H). ¹³CNMR (151 MHz, DMSO-d6) δ 159.10, 152.91, 150.91, 136.04, 133.96, 131.15,129.93, 129.59, 129.13, 128.54, 128.51, 127.28, 127.03, 126.03, 118.94,116.34, 106.07, 101.81, 31.48, 27.47, 20.97. MS (ES+) (m/z): [M+1]⁺calculated for C₂₁H₁₉N₃O₃, 362.15. found 362.09.

6-(5-Chlorofuran-2-yl)-7,9-dimethyl-11-(m-tolyl)-6Hbenzo[b]pyrimido[4′,5′-3,4]pyrrolo[1,2-d]oxazine-8,10-(7H,9H)-dione(BPO-19). 5-chlorofurfural (43 mg, 329 μmol), 5d (100 mg, 277 μmol), asmall crystal of TsOH, and 1,2-dichloroethane (2.5 mL) were placed in anEmrys 2-5 mL process vial and submerged to the level of solvent in anoil bath at 150° C. for 20 min. Once the reaction had cooled thecontents were purified by TLC prep to give BPO-19 (88 mg, 67%) as an offwhite solid. ¹H NMR (600 MHz, CD₂Cl₂) δ 7.59 (s, 0.5H), 7.56 (d, J=7.6,0.5H), 7.46 (t, J=7.6, 0.5H), 7.30 (d, J=7.7, 1H), 7.21 (t, J=7.6,0.5H), 7.06-6.98 (m, 2H), 6.89 (s, 0.5H), 6.85 (d, J=7.6, 0.5H), 6.77(s, 1H), 6.65 (qd, J=2.3, 6.3, 1H), 6.54 (dd, J=8.2, 12.6, 1H), 6.00 (s,2H), 3.47 (s, 3H), 3.25 (d, J=6.6, 3H), 2.47 (s, 1.5 H), 2.23 (s, 1.5H).¹³C NMR (151 MHz, CD₂Cl₂) δ 159.33, 151.85, 150.01, 145.39, 145.34,138.58, 138.43, 138.10, 132.23, 131.16, 131.13, 130.32, 130.29, 130.17,130.14, 128.80, 128.52, 128.38, 127.07, 127.05, 126.92, 125.93, 124.30,122.55, 122.50, 120.37, 120.33, 119.64, 114.40, 107.31, 106.26, 106.21,105.55, 105.49, 68.10, 32.40, 32.37, 27.94, 27.92, 21.64, 21.35. MS(ES+) (m/z): [M+1]⁺ calculated for C₂₆H₂₁ClN₃O₄, 474.12. found 474.02.

Example 23 Synthesis of BPO-20

7,9-Dimethyl-6-(5-methylfuran-2-yl)-11-(m-tolyl)-6H-benzo[b]pyrimido[4′,5′-3,4]pyrrolo[1,2-d]oxazine-8,10-(7H,9H)-dione(BPO-20).

5-Methylfurfural (33 μL, 37 mg, 332 mol), 5d (100 mg, 277 mol) (seeExample 22 for synthesis of 5d), TFA (5 μL, 7.4 mg, 65 μmol), andchloroform (3 mL) were sealed in a Emrys 2-5 mL process vial andsubmerged to the level of solvent in an oil bath at 150° C. for 24 min.Once the reaction had cooled, the contents were purified by flashchromatography to give BPO-20 (92 mg, 74%) with a trace amount ofimpurity. The product was dissolved in a minimum volume of CH₂Cl₂ andthen diluted with methanol (6 mL). The solution was placed on a rotaryevaporator until a precipitate began to form. The mixture was thencooled, the precipitate filtered and washed with cold methanol to giveBPO-20 (64 mg, 51%) as a pure ivory colored powder. ¹H NMR (600 MHz,CD₂Cl₂) δ 7.60 (s, 0.5H), 7.57 (d, J=7.6, 0.5H), 7.47 (t, J=7.6, 0.5H),7.30 (d, J=7.7, 1H), 7.21 (t, J=7.6, 0.5H), 7.05-6.97 (m, 2H), 6.91 (s,0.5H), 6.86 (d, J=7.6, 0.5H), 6.77 (s, 1H), 6.67-6.60 (m, 1H), 6.55 (dd,J=8.3, 11.3, 1H), 5.85 (d, J=2.8, 1H), 5.77 (d, J=2.5, 1H), 3.47 (s,3H), 3.26 (d, J=6.2, 3H), 2.48 (s, 1.5H), 2.23 (s, 1.5H), 2.23 (s, 3H).¹³C NMR (151 MHz, CD₂Cl₂) δ 159.44, 159.43, 154.49, 151.89, 148.66,145.72, 145.67, 138.52, 138.37, 132.28, 130.75, 130.72, 130.38, 130.33,130.31, 130.19, 128.84, 128.48, 128.34, 126.98, 126.89, 126.87, 126.08,123.82, 122.27, 122.22, 120.34, 120.30, 119.59, 113.01, 107.52, 107.47,106.40, 105.49, 105.42, 68.53, 32.37, 32.34, 27.91, 27.89, 21.64, 21.35,13.81. MS (ES+) (m/z): [M+1]⁺ calculated for C₂₇H₂₄N₃O₄, 454.18. found454.10.

Example 24 Synthesis of BPO-21

6-(2-hydroxy-5-nitrophenyl)-1,3-dimethyl-5-phenyl-1Hpyrrolo[3,4-d]pyrimidine-2,4(3H,6H)-dione(5g). In a 25 mL round bottom flask was placed 4a (1.00 g, 2.97 mmol)(see Example 2 for synthesis of 4a), 2-amino-4-nitrophenol (921 mg, 5.98mmol), and methanol (12 mL). The reaction was stirred at reflux for 5 h,then cooled in an ice bath. The precipitate was filtered and rinsed withcold methanol to give 5g (1.32 g, 94%). ¹H NMR (600 MHz, DMSO-d6) δ11.72 (s, 1H), 8.19 (d, J=2.8, 1H), 8.15 (dd, J=2.9, 9.1, 1H), 7.31-7.21(m, 5H), 7.08 (s, 1H), 7.01 (d, J=9.1, 1H), 3.34 (s, 3H), 3.21 (s, 3H).¹³C NMR (151 MHz, DMSO-d6) δ 159.26, 159.03, 150.85, 138.89, 134.18,130.34, 129.34, 128.85, 128.14, 127.36, 126.15, 126.09, 125.45, 116.60,105.95, 102.41, 31.52, 27.48. MS (ES+) (m/z): [M+1]⁺ calculated forC₂₀H₁₇N₄O₅, 393.12. found 393.02.

6-(5-bromofuran-2-yl)-7,9-Dimethyl-2-nitro-11-phenyl-6H-benzo[b]pyrimido[4′,5′-3,4]pyrrolo[1,2-d]oxazine-8,10-(7H,9H)-dione(BP0-21).

Pyrrole 5g (225 mg, 573 μmol), 5-bromofurfural (130 mg, 743 μmol),1,2-dicholoroethane (4 mL), TFA (10 μL, 14.9 mg, 130 mol) and 3 Åmolecular sieve (500 mg, 8-12 mesh) were sealed in a Emrys 2-5 mLprocess vial and submerged to the level of solvent in an oil bath at150° C. for 100 min. Upon cooling the reaction turned dark green andsome impurities precipitated. The reaction was filtered through a celiteplug and dried in vacuo. The residue was dissolved in CH₂Cl₂ (3 mL) andthen diluted with ethanol (15 mL). The solution was then placed on arotary evaporator until small crystals began to form. The mixture wascooled and the crystals were collected by filtration and rinsed withcold ethanol to give BPO-21 (204 mg, 65%). ¹H NMR (600 MHz, CD₂Cl₂) δ7.91 (dd, J=2.6, 8.9, 1H), 7.84 (d, J=7.7, 1H), 7.69 (t, J=7.6, 1H),7.58 (t, J=7.5, 1H), 7.39 (d, J=2.6, 1H), 7.37 (t, J=7.6, 1H), 7.18 (d,J=8.9, 1H), 7.07 (d, J=7.7, 1H), 6.92 (s, 1H), 6.17 (d, J=3.4, 1H), 6.03(d, J=3.4, 1H), 3.48 (s, 4H), 3.27 (s, 3H). ¹³C NMR (151 MHz, CD₂Cl₂) δ159.10, 151.70, 151.31, 150.71, 142.06, 131.75, 131.63, 130.16, 129.53,129.32, 129.18, 129.07, 125.55, 124.83, 124.74, 122.43, 120.23, 115.93,115.22, 112.56, 106.48, 105.26, 68.75, 32.40, 28.01. MS (ES+) (m/z):[M+1]⁺ calculated for C₂₅H₁₈BrN₄O₆, 549.04. found 549.05.

Example 25 Synthesis of BPO-22

7,9-Dimethyl-6-(5-methylfuran-2-yl)-2-nitro-11-phenyl-6H-benzo[b]pyrimido[4′,5′-3,4]pyrrolo[1,2-d]oxazine-8,10-(7H,9H)-dione(BPO-22).

Pyrrole 5g (250 mg, 637 μmol) (see Example 24 for synthesis of 5g),5-methylfurfural (76 μl, 84 mg, 764 μmol), chloroform (8 mL), and TFA(10 pt, 14.9 mg, 130 μmol) were sealed in a Emrys 2-5 mL process vialand submerged to the level of solvent in an oil bath at 150° C. for 2.7h. The reaction was then cooled and dried in vacuo. The residue waspurified by flash chromatography to give BPO-22 (245 mg, 79%) as ayellow foam after drying under high vacuum. To convert the foam to apowder, the foam was dissolved in CH₂Cl₂ (3 mL) and then diluted withmethanol (15 mL). The volume of solvent was then reduced on a rotaryevaporator. Once the solution began to precipitate the mixture wasallowed to stand until precipitation ceased. The mixture was thencooled, filtered, and the precipitate rinsed with cold methanol to giveBPO-22 (179 mg). The mother liquor was reduced to yield additionalBPO-22 (38 mg). ¹H NMR (600 MHz, CD₂Cl₂) δ 7.89 (dd, J=2.6, 8.9, 1H),7.84 (d, J=7.7, 1H), 7.68 (t, J=7.6, 1H), 7.57 (t, J=7.5, 1H), 7.39 (d,J=2.4, 1H), 7.36 (t, J=7.6, 1H), 7.14 (d, J=8.9, 1H), 7.07 (d, J=7.7,1H), 6.90 (s, 1H), 5.91 (d, J=3.1, 1H), 5.79 (d, J=2.9, 1H), 3.48 (s,3H), 3.26 (s, 3H), 2.20 (s, 3H). ¹³C NMR (151 MHz, CD₂Cl₂) δ 159.20,155.08, 151.75, 151.18, 147.73, 141.85, 131.80, 131.20, 130.05, 129.59,129.51, 129.14, 129.02, 125.71, 124.23, 122.28, 120.11, 115.90, 113.51,106.62, 106.51, 106.41, 69.32, 32.37, 27.98, 13.81. MS (ES+) (m/z):[M+1]⁺ calculated for C₂₆H₂₁N₄O₆, 485.15. found 485.03

Example 26 Synthesis of PPQ-23

6-(2-Amino-5-nitrophenyl)-1,3-dimethyl-5-phenyl-1H-pyrrolo[3,4-d]pyrimidine-2,4(3H,6H)-dione(5b). In a 25 mL round bottom flask 4-nitro 1,2-phenylenediamine (300mg, 1.96 mmol), and 4a (330 mg, 979 mmol) (see Example 2 for synthesisof 4a) were combined in ethanol (10 mL). The stirred mixture was broughtto reflux for 3 h during which a yellow precipitate formed. The mixturewas then chilled in an ice bath, filtered, and the precipitate washedwith cold ethanol to give 5b (340 mg, 88.7%) as a waxy shiny yellowsolid; mp>300° C. ¹H NMR (600 MHz, DMSO-d6) δ 7.97 (dd, J=2.7, 9.2, 1H),7.86 (d, J=2.6, 1H), 7.37-7.32 (m, 2H), 7.28-7.24 (m, 4H), 7.05 (s, 1H),6.75 (d, J=9.2, 1H), 6.65 (s, 2H), 3.35 (s, 3H), 3.21 (s, 3H). ¹³C NMR(151 MHz, DMSO-d6) δ 159.06, 151.66, 150.89, 135.01, 133.84, 130.38,129.34, 129.02; 128.32, 127.38, 126.19, 126.05, 121.34, 114.31, 105.49,102.98, 31.59, 27.46. MS (ES+) (m/z): [M+1]⁺ calculated for C₂₀H₁₈N₅O₄,392.135880. found 392.03.

6-(5-Bromofuran-2-yl)-7,9-dimethyl-2-nitro-11-phenyl-5,6-dihydropyrimido[4′,5′-3,4]pyrrolo[1,2-a]quinoxaline-8,10-(7H,9H)-dione(PPQ-23). In a 5 mL pear shaped flask was placed 5b (100 mg, 256 umol),5-bromo-2-furaldehyde (50 mg, 286 μmol), TFA (10 μL, 14.9 mg, 130 μmol),and chloroform (2.5 mL). The mixture was refluxed for 1 h, dried invacuo, and purified by flash chromatography. The desired fractions werecombined and dried in vacuo. The remaining solid was triturated in hotbenzene and filtered to give PPQ-23 (103 mg, 73.6%) as a bright yellowsolid. ¹H NMR (600 MHz, CD₂Cl₂) δ 7.84 (d, J=7.7, 1H), 7.82 (dd, J=2.4,8.9, 1H), 7.66 (t, J=7.3, 1H), 7.53 (t, J=7.5, 1H), 7.37 (d, J=2.3, 1H),7.33 (t, J=7.6, 1H), 7.02 (d, J=7.7, 1H), 6.89 (d, J=8.9, 1H), 6.16 (d,J=2.3, 1H), 6.15 (d, J=3.4, 1H), 5.90 (dd, J=0.8, 3.4, 1H), 5.71 (d,J=2.5, 1H), 3.54 (s, 3H), 3.28 (s, 3H). ¹³C NMR (151 MHz, CD₂Cl₂) δ159.31, 154.54, 151.89, 142.72, 139.43, 131.86, 131.25, 130.03, 129.79,129.55, 129.05, 128.93, 123.95, 123.30, 122.99, 122.41, 116.84, 116.62,112.38, 111.97, 107.73, 106.20, 48.22, 32.03, 28.02. MS (ES+) (m/z):[M+1]⁺ calculated for C₂₅H₂₀BrN₄O₃, 548.06. found 547.96.

Example 27 Synthesis of BPO-24

6-(5-Chloro-2-hydroxy-4-nitrophenyl)-1,3-dimethyl-5-phenyl-1H-pyrrolo[3,4-d]pyrimidine-2,4(3H,6H)-dione(5f). In a 10 mL round bottom flask was placed 4a (200 mg, 595 mmol)(see Example 2 for synthesis of 4a), 2-amino-4-chloro-5-nitrophenol (236mg, 1.25 mmol), and ethanol (5 mL). The reaction was stirred at refluxfor 5 h, then cooled and placed in a freezer. After several hours yellowcrystals formed, which were filtered and rinsed with cold ethanol togive 5f (225 mg, 89%). ¹H NMR (600 MHz, DMSO-d6) δ 11.33 (s, 1H), 7.79(s, 1H), 7.44 (s, 1H), 7.30 (s, 5H), 7.10 (s, 1H), 3.33 (s, 3H), 3.20(s, 3H). ¹³C NMR (151 MHz, DMSO-d6) δ 158.98, 152.39, 150.82, 147.31,134.14, 131.79, 130.50, 130.32, 129.23, 128.96, 128.33, 127.48, 113.90,112.93, 105.91, 102.66, 31.55, 27.51. MS (ES+) (m/z): [M+1]⁺ calculatedfor C₂₀H₁₆ClN₄O₅, 427.08. found 427.12.

2-Chloro-7,9-Dimethyl-6-(5-methylfuran-2-yl)-3-nitro-11-phenyl-6H-benzo[b]pyrimido[4′,5′-3,4]pyrrolo[1,2-d]oxazine-8,10-(7H,9H)-dione(BPO-24).

Pyrrole 5f (97 mg, 227 μmol), 5-methylfurfural (25 μl, 27.7 mg, 252μmol), TFA (5 μL, 7.4 mg, 65 mmol), 1,2-dichloroethane (2 mL) and a stirbar were sealed in a Emrys 2-5 mL process vial and submerged to thelevel of solvent in an oil bath at 150° C. for 2 h. The reaction wasthen cooled, diluted with ethanol (8 mL), and placed in a freezer. Afterseveral hours a precipitate formed, which was filtered and rinsed withcold ethanol to give BPO-24 (80 mg, 68%). ¹H NMR (600 MHz, CD₂Cl₂) δ7.80 (d, J=7.7, 1H), 7.64 (dd, J=6.1, 7.0, 2H), 7.58 (t, J=7.5, 1H),7.42 (t, J=7.2, 1H), 7.08 (d, J=7.7, 1H), 6.87 (s, 1H), 6.58 (s, 1H),5.92 (d, J=3.2, 1H), 5.82 (d, J=2.3, 1H), 3.47 (s, 3H), 3.26 (s, 3H),2.23 (s, 3H). ¹³C NMR (151 MHz, CD₂Cl₂) δ 159.06, 155.28, 151.65,147.36, 144.47, 144.05, 131.75, 131.54, 130.24, 130.02, 129.76, 129.15,129.12, 128.96, 124.45, 122.71, 120.59, 117.46, 113.80, 107.06, 106.79,106.71, 69.18, 32.36, 28.02, 13.84. MS (ES+) (m/z): [M+1]⁺ calculatedfor C₂₆H₂₀ClN₄O₆, 519.11. found 519.14.

Example 28 Synthesis of BPO-25

Ethyl6-(5-Bromofuran-2-yl)-7,9-dimethyl-8,10-dioxo-11-phenyl-7,8,9,10-tetrahydro-6H-benzo[b]pyrimido[4′,5′:3,4]pyrrolo[1,2-d][1,4]oxazine-2-carboxylate(BPO-25). (See also Example 2). Pyrrole 5h (500 mg, 1.19 mmol),5-bromofurfural (240 mg, 1.37 mmol), chloroform (7 mL), TFA (10 μL, 14.8mg, 130 mol), and 3 Å molecular sieves (2.0 g, 8-12 mesh beads) weresealed in an Emrys 10-20 mL process vial and submerged to the level ofsolvent in an oil bath at 150° C. (An Emrys process vial is acommercially available thick walled vessel much like a test tubetypically used for microwave reactions that may be sealed using adisposable plastic-lined metal cap. Precautions should be taken becausethe reaction vessel is under pressure when heated.) The reaction wasstirred for 24 min then removed from the oil bath. Once the internalpressure had dropped the reaction vial was rapidly cooled in water.After cooling, the reaction was filtered through celite into a 50 mLrecovery flask and the dried in vacuo. The residue was dissolved in aminimum amount of CH₂Cl₂ and quickly diluted with warm ethanol (25 mL).Fine crystals began to form immediately. The mixture was then placed ona rotary evaporator and the CH₂Cl₂ was removed to increase the quantityof crystals. The mixture was then chilled, filtered, and the crystalsrinsed with cold ethanol to give BPO-25 (0.500 g, 76.4%) as fine whiteneedle like crystals. No m.p. (slow decomposition). ¹H NMR (600 MHz,CD₂Cl₂) δ 7.81 (d, J=7.7, 1H), 7.68 (dd, J=1.9, 8.4, 1H), 7.63 (t,J=7.5, 1H), 7.52 (t, J=7.5, 1H), 7.34 (t, J=7.5, 1H), 7.23 (d, J=1.8,1H), 7.09 (d, J=8.4, 1H), 7.06 (d, J=7.7, 1H), 6.86 (s, 1H), 6.14 (d,J=3.4, 1H), 5.98 (d, J=2.9, 1H), 4.11 (dq, J=7.2, 10.7, 1H), 4.00 (dq,J=7.1, 10.7, 1H), 3.48 (s, 3H), 3.26 (s, 3H), 1.14 (t, J=7.1, 3H). ¹³CNMR (151 MHz, CD₂Cl₂) δ 165.03, 159.26, 151.87, 151.79, 149.08, 131.78,131.27, 130.00, 129.80, 129.68, 128.84, 128.81, 128.29, 125.40, 124.69,124.47, 124.42, 121.57, 119.64, 114.91, 112.43, 105.93, 105.67, 68.40,61.28, 32.38, 27.95, 14.20. MS (ES+) (m/z): [M+1]⁺ calculated forC₂₈H₂₃BrN₃O₆, 576.08. found 576.04.

Example 29 Synthesis of BPO-26

Ethyl7,9-Dimethyl-8,10-dioxo-6-(5-methylfuran-2-yl)-11-phenyl-7,8,9,10-tetrahydro-6H-benzo[b]pyrimido[4′,5′:3,4]pyrrolo[1,2-d][1,4]oxazine-2-carboxylate(BPO-26). Pyrrole 5h (138 mg, 329 μmol) (see Example 2 for synthesis of5h), 5-methylfurfural (36 μL, 39.9 mg, 362 μmol), chloroform (3 mL), andTFA (5 μL, 7.4 mg, 65 μmol) sealed in a Emrys 2-5 mL process vial andsubmerged in an oil bath at 150° C. for 2 h. After cooling the reactionwas dried in vacuo. The residue was dissolved in a minimum volume ofCH₂Cl₂ and then diluted with ethanol (10 mL). The solution was thenplaced on a rotary evaporator and solvent removed until the mixtureceased to precipitate. The mixture was then chilled, filtered, and theprecipitate rinsed with cold ethanol to give BPO-26 (168 mg, 82%). ¹HNMR (600 MHz, CD₂Cl₂) δ 7.81 (d, J=7.7, 1H), 7.67 (dd, J=1.9, 8.4, 1H),7.63 (t, J=7.5, 1H), 7.52 (t, J=7.5, 1H), 7.34 (t, J=7.5, 1H), 7.23 (d,J=1.8, 1H), 7.09-7.02 (m, J=4.5, 8.0, 2H), 6.83 (s, 1H), 5.85 (d, J=3.2,1H), 5.76 (d, J=2.5, 1H), 4.10 (dq, J=7.1, 10.7, 1H), 3.99 (dq, J=7.1,10.7, 1H), 3.48 (s, 3H), 3.26 (s, 3H), 2.21 (s, 3H), 1.14 (t, J=7.1,3H). ¹³C NMR (151 MHz, CD₂Cl₂) δ 165.10, 159.37, 154.76, 151.83, 149.47,148.20, 131.81, 130.84, 130.16, 129.72, 129.70, 128.80, 128.76, 128.13,125.54, 124.38, 123.96, 121.54, 119.56, 113.22, 106.89, 106.49, 105.84,68.89, 61.22, 32.34, 27.92, 14.20, 13.80. MS (ES+) (m/z): [M+1]⁺calculated for C₂₉H₂₆N₃O₆, 512.18. found 512.22.

Example 30 Biological Techniques and Methods

Cell Culture and Plate Reader Assay of CFTR Inhibition—Fischer ratthyroid (FRT) cells co-expressing human wild type CFTR and the halideindicator YFP-H148Q were cultured in 96-well black-walled microplates(Corning Costar) at a density of 20,000 cells per well in Coon'smodified F12 medium containing 10% fetal bovine serum, 2 mM L-glutamine,100 U/mL penicillin and 100 μg/mL streptomycin. CFTR chlorideconductance was assayed at 48 h after plating on a FluoStar fluorescenceplate reader (BMG Lab Technologies) as described (see, e.g., Ma et al.,J Clin Invest. 2002, supra). Each well was washed 3 times with PBS,leaving 60 μL PBS. Test compounds were added and incubated with thecells for 45 min. Then, 5 μL of a CFTR-activating cocktail (10 μMforskolin, 100 μM IBMX, 20 μM apigenin in PBS) was added. After 15 min,each well was assayed for iodide influx by recording fluorescencecontinuously (200 ms per point) for 2 s (baseline) and then for 10 safter rapid addition of 160 μL of isosmolar PBS in which 137 mM chloridewas replaced by iodide. The initial rate of iodide influx was computedfrom fluorescence data by non-linear regression.

Short-Circuit Current—Snapwell inserts containing CFTR-expressing FRTcells were mounted in an Ussing chamber. The hemichambers contained 5 mLof buffer containing 75 mM NaCl and 75 mM Na gluconate (apical) and 150mM NaCl (basolateral) (pH 7.3), and the basolateral membrane waspermeabilized with 250 μg/mL amphotericin B, as described (see, e.g.,Sonawane et al., FASEB J 2006, supra). Short-circuit current wasrecorded continuously using a DVC-1000 voltage clamp (World PrecisionInstruments) using Ag/AgCl electrodes and 3 M KCl agar bridges.

In Vitro Metabolism in Hepatic Microsomes—A compound of structure (I)was incubated for specified times at 37° C. with rat liver microsomes(e.g., 1 mg protein/ml; Sigma-Aldrich, St. Louis, Mo.) in potassiumphosphate buffer (100 mM) containing NADPH (0 or 1 mM). The mixture wasthen chilled on ice, and 0.5 ml of ice-cold ethyl acetate was added.Samples were centrifuged for 15 min at 3,000 rpm, and the supernatantwas evaporated to dryness under nitrogen. The residue was dissolved in150 μl mobile phase (acetonitrile:water 3:1, containing 0.1% formicacid) for LC/MS analysis. Reverse-phase HPLC separations were carriedout using a Waters C18 column (2.1×100 mm, 3.5 mm particle size)equipped with a solvent delivery system (Waters model 2690, Milford,Mass.). The solvent system consisted of a linear gradient from 5 to 95%acetonitrile run over 16 min (0.2 mL/min flow rate). Mass spectra wereacquired on an Alliance HT 2790+ZQ mass spectrometer using negative iondetection.

Mouse Pharmacokinetics and Renal Accumulation—A compound of structure(I) was formulated at 1 mg/mL in 5% DMSO, 2.5% Tween-80 and 2.5% PEG400in H₂O, based on formulations used for compounds of similar polarity andchemical properties. Male mice in a CD1 genetic background (age 8-10weeks, 25-35 g) were administered 300 μL of the BPO-27 formulation byintraperitoneal injection. At specified times kidneys were removedfollowing renal arterial perfusion with PBS. Kidneys were weighed, mixedwith acetic acid (100 μl per 1 g tissue) and ethyl acetate (10 mL per 1g tissue), and homogenized. The homogenate was centrifuged at 3,000 rpmfor 15 min. Calibration standards were prepared in kidney homogenatesfrom control mice to which was added known amounts of BPO-27. The ethylacetate-containing supernatant was dried under nitrogen and the residuewas reconstituted in acetonitrile:H₂O (3:1) containing 0.1% formic acid.For analysis of blood and urine, fluids were diluted with equal volumeof water and extracted with ethyl acetate. LC/MS analysis was carriedout as described herein.

Liquid Chromatography/Mass Spectrometry—Compounds (each at 5 μM) wereincubated for specified times at 37° C. with rat liver microsomes (1 mgprotein/ml; Sigma-Aldrich, St. Louis, Mo.) in potassium phosphate buffer(100 mM) containing NADPH (0 or 1 mM). The mixture was then chilled onice, and 0.5 ml of ice-cold ethyl acetate was added. Samples werecentrifuged for 15 min at 3,000 rpm, and the supernatant was evaporatedto dryness under nitrogen. The residue was dissolved in 150 mobile phase(acetonitrile:water 3:1, containing 0.1% formic acid) for LC/MSanalysis. Reverse-phase HPLC separations were carried out using a WatersC18 column (2.1×100 mm, 3.5 mm particle size) equipped with a solventdelivery system (Waters model 2690, Milford, Mass.). The solvent systemconsisted of a linear gradient from 5 to 95% acetonitrile run over 16min (0.2 mL/min flow rate). Mass spectra were acquired on an Alliance HT2790+ZQ mass spectrometer using negative ion detection, scanning from150 to 1500 Da. The electrospray ion source parameters were as follows:capillary voltage 3.5 kV (positive ion mode), cone voltage 37 V, sourcetemperature 120° C., desolvation temperature 250° C., cone gas flow 25L/h, and dessolvation gas flow 350 L/h.

Embryonic Organ Culture Model of PKD—Mouse embryos were obtained atembryonic day 13.5 (E13.5). Metanephroi were dissected and placed ontransparent Falcon 0.4-mm diameter porous cell culture inserts asdescribed (see, e.g., Tradtrantip et al., J. Med. Chem. 2009, supra). Tothe culture inserts was added DMEM/Ham's F-12 nutrient mediumsupplemented with 2 mM L-glutamine, 10 mM HEPES, 5 μg/mL insulin, 5μg/mL transferrin, 2.8 nM selenium, 25 ng/ml prostaglandin E, 32 pg/mlT3, 250 U/ml penicillin, and 250 μg/ml streptomycin. Kidneys weremaintained in a 37° C. humidified CO₂ incubator for up to 8 days.Culture medium containing 100 μM 8-Br-cAMP, with or without testcompound, was replaced (in the lower chamber) every 12 h. Kidneys werephotographed using a Nikon inverted microscope (Nikon TE 2000-S)equipped with 2× objective lens, 520 nm bandpass filter, andhigh-resolution CCD camera. Percentage cyst area was calculated as totalcyst area divided by total kidney area.

Example 31 Identification and Characterization of PPQ Analogs

PPQ-102 is a small-molecule inhibitor of CFTR chloride conductance withefficacy in preventing and reversing cyst formation in an organ culturemodel of PKD (see, e.g., Tradtrantip et al., J. Med. Chem. 2009, supra;Int'l Patent Appl. Publication No. WO 2011/019737). Preliminary analysisof the metabolic stability and aqueous solubility of PPQ-102, indicatedpoorer metabolic stability and lower water solubility (˜2 μM inalbumin-free saline) than desired for optimal drugability. In vitrometabolic stability was determined by compound incubation with hepaticmicrosomes at 37° C. for specified times in the absence vs. presence ofNADPH, following by LC/MS analysis. Shown in FIG. 6A is PPQ-102disappearance in hepatic microsomes in the presence of NADPH, with ˜60%disappearance in 30 min.

No loss of PPQ-102 was seen in the absence of NADPH. PPQ-102 wasundetectable in serum, kidney, and urine at 30-60 min after intravenousbolus administration of 300 μg PPQ-102 in mice using an LC/MS assay withsensitivity better than 100 nM. Though the precise metabolic fate ofPPQ-102 is not known, structural considerations and the presence ofprominent metabolites at +14 and +16 daltons (see FIG. 6B) suggestedpossible oxidation, aromatization, and hydroxylation (see FIG. 6C). Toimprove on the drug-like properties of PPQ-102, a series of compoundanalogs were synthesized and tested.

Initial testing of the compounds for the capability to inhibit CFTR wasperformed using a plate-reader assay in which iodide influx was measuredin FRT cells co-expressing human wild type CFTR and the iodide-sensingyellow fluorescent protein YFP-H148Q/1152L. FIG. 7A (top) showsrepresentative fluorescence data for inhibition of CFTR-mediated iodideinflux by one of the synthesized PPQ analogs, which had a reducednegative slope after iodide addition with increasing inhibitionconcentration. FIG. 7A (bottom) shows concentration-inhibition data forselected compounds. Table B below provides IC₅₀ values for PPQ and BPOcompounds.

TABLE B CFTR inhibition of BPO and PPQ analogs.

Compound X Y R¹ R² R³ R⁴ R⁵ R⁶ IC₅₀ (μm)^(A) IC₅₀ (μM)^(B) PPQ-102 ON(R⁴) H H H H H Me 0.25 0.1 PPQ-1 O N(R⁴) H H H CH₃SO₂ H Me inactivePPQ-2 O N(R⁴) H H H ClCH₂CO₂ H Me inactive PPQ-3 O N(R⁴) H H H CH₃CO₂ HMe inactive PPQ-4 O N(R⁴) H H H NO H Me 2 PPQ-5 O N(R⁴) H H H Me H Me 1PPQ-6 O N(R⁴) H H H H H Et 0.3 PPQ-7 S N(R⁴) H H H H H Me 1.7 PPQ-8 ON(R⁴) H H H H Me Me 1.7 PPQ-9 O N(R⁴) H H H H H Cl 0.15 PPQ-10 O N(R⁴) HH H H H Br 0.09 0.05 PPQ-11 O N(R⁴) H H H H H I 0.17 0.1 PPQ-12 O N(R⁴)H H H H H CF₃ 0.35 PPQ-13 O N(R⁴) H H H H H Ph inactive PPQ-14 O N(R⁴) HH H H H CH₂OH inactive PPQ-15 O N(R⁴) H H H H —(CH)₄— inactive BPO-16 OO H H H H Br 0.2 0.09 BPO-17 O O H H H H I 0.5 BPO-18 O O H H H H Me0.15 BPO-19 O O (m-)Me H H H Cl 0.37 BPO-20 O O (m-)Me H H H Me 0.6BPO-21 O O H NO₂ H H Br 0.12 0.025 BPO-22 O O H NO₂ H H Me 0.1 0.025PPQ-23 O N(R⁴) H NO₂ H H H Br 0.17 BPO-24 O O H Cl NO₂ H Me 0.17 BPO-25O O H COOEt H H Br 0.05 0.025 BPO-26 O O H COOEt H H Me 0.08 BPO-27 O OH COOH H H Br 0.04 0.008 (R)-BPO-27 O O H COOH H H Br — 0.004^(A)Determined by micro-plate reader assay ^(B)Determined byshort-circuit current assay

The relative IC₅₀ values obtained from the plate reader assay are usefulfor comparisons. Absolute IC₅₀ values obtained from the plate-readerassay are approximate, generally underestimating compound potencybecause of assay non-linearities, pH-dependent YFP fluorescence, the useof iodide instead of chloride, and compound dilution at the start of theassay (see, e.g., Galietta et al., Am. J. Physiol. Cell Physiol.281:C1734-42 (2001)).

Quantitative concentration-inhibition data for the most potent compoundswere obtained by analysis of short-circuit current, which represents adefinitive electrophysiological measure of compound potency. Current wasmeasured in CFTR-expressing FRT cells in which the basolateral membranewas permeabilized and in the presence of a transepithelial chlorideconcentration gradient, so that current is a quantitative linear measureof CFTR function. CFTR was activated by forskolin, followed by serialadditions of increasing concentrations of test compounds. FIG. 7Billustrates a representative short-circuit current measurement.Increased CFTR chloride conductance was observed following addition thecAMP agonist forskolin. Conductance was reduced in aconcentration-dependent manner by a PPQ inhibitor, with completeinhibition observed at high inhibitor concentration.

Initial synthesis efforts focused on preventing aromatization, which wasinitially achieved by derivatizing the secondary amine of PPQ-102 togive analogs PPQ-(1-5). The PPQ-102-derived amides PPQ-(1-3) were foundto be substantially less active than PPQ-102. Because the loss of thebasic amine coincided with a loss in activity, nitrosamine PPQ-4 wassynthesized, but attempts to reduce the nitrosamine to a strongly basichydrazine only yielded PPQ-102. To maintain basicity and to preventaromatization, synthesis of the N-methyl analog PPQ-5 was attempted viareductive alkylation using formaldehyde, but these efforts wereunsuccessful. However, the N-methyl precursor 5c was obtained in goodyield from N-methyl-1,2-phenylenediamine and 4a. Pyrrole 5c was thenused to synthesize PPQ-5, which was weakly active.

Re-examination of SAR data obtained from ˜350 commercially availableanalogs (see, e.g., Tradtrantip et al., J. Med. Chem. 2009, supra; Int'lPatent Appl. Publication No. WO 2011/019737) suggested that the5-position of the furan ring in PPQ-102 was privileged. Therefore,compounds PPQ-(6-15) were synthesized to probe the steric and electronicrequirements for activity of the furyl moiety. Substituting bromine atthe 5-position of the furyl ring yielded PPQ-10, which was substantiallymore stable than PPQ-102 and had similar CFTR inhibition potency (seeFIGS. 5B, 5C). To further probe oxidative aromatization as a metabolicpathway, the secondary amine of PPQ-10 was replaced by oxygen forming anether bridge, which is unable to undergo oxidation. The resultingbenzoxazine BPO-16 had similar CFTR inhibition potency (see FIG. 7A) andbetter stability than PPQ-102. Synthesis of BPO-(17-18) confirmed CFTRinhibition activity (see Table B) and improved stability (see FIG. 7C)imparted by bromine and illustrated the synergy between the 5-bromofuranmoiety and the ether bridge.

Previous SAR data (see, e.g., Tradtrantip et al., J. Med. Chem. 2009,supra) showed that an m-tolyl moiety (R¹=Me) increased CFTR inhibitionpotency. However, synthesis of BPO-(19-20) revealed that the m-tolylmoiety reduced CFTR inhibition in the BPO series. To increase thepolarity and hence the aqueous solubility of BPO-16, a nitro group wasintroduced at R². The resulting compound, BPO-21, had substantiallygreater CFTR inhibition potency (see FIG. 8A) with excellent metabolicstability in hepatic microsomes (see FIG. 8B). BPO-22 and PPQ-23 showedthat the increased CFTR inhibition activity conferred by the nitrosubstituent at R² was independent of both the 5-bromo substituent andthe ether bridge.

To increase compound polarity further, the nitro functionality at R₂ inBPO-21 was replaced with a carboxyl moiety to provide BPO-27. BPO-27 wasthe most potent CFTR inhibitor with IC₅₀˜8 nM (see FIG. 8A), and hadexcellent stability in hepatic microsomes with <5% compound loss in 30min (see FIG. 8B). At physiological pH BPO-27 is deprotonated and thussubstantially more polar then PPQ-102 (clogP 1.76 for BPO-27 vs.clogP4.92 for PPQ-102) and with solubility of 17 μM in a pH 7.4 aqueousphosphate buffer. BPO-27 was synthesized by hydrolysis of the ethylester BPO-25, which, interestingly, also had excellent CFTR inhibitionactivity, and could potentially serve as a pro-drug of BPO-27, tofacilitate efficient intestinal absorption and cell accumulationfollowing de-esterification by ubiquitous intracellular esterases.

SAR analysis suggests that the relative stability of BPO-27 comparedwith PPQ-102 is the consequence of the 5-Br substituted furan and theether bridge, which largely prevent hydroxylation and aromatizationmodifications. The greatly improved water solubility of BPO-27 comparedwith PPQ-102 is a consequence of the carboxylic acid substituent, whichis charged at physiological pH. The carboxylic acid addition also,unexpectedly, improved CFTR inhibition potency.

Example 32 Reduction of Cyst Growth in a PKD Kidney Organ Culture Model

An established embryonic ex vivo kidney organ culture model of PKD wasused to test BPO-27 (racemic mixture of R and S forms) efficacy inreducing cAMP agonist-induced renal cystogenesis (see Example 30).Progressive renal cyst formation and growth in 8-Br-cAMP agonist-treatedcultures, as seen by transmitted light microscopy, is shown in FIG. 9A.Kidney growth without cyst formation was observed in the absence of8-Br-cAMP. Cyst growth in the 8-Br-cAMP-treated kidney was remarkablyreduced by inclusion of BPO-27 in the culture medium. As quantified bypercentage area occupied by cysts, BPO-27 inhibited cyst growth withIC₅₀ of approximately 100 nM (see FIG. 9B), much better than thatof >500 nM measured for PPQ-102 (see, e.g., Tradtrantip et al., J. Med.Chem. 2009, supra). Thus, racemic BPO-27 was shown to have prevented andreversed renal cyst formation in an embryonic kidney culture model ofPKD.

Example 33 CFTR Inhibition of Isolated Enantiomers of BPO-27

The racemic mixture of BPO-27 was further separated into its respectiveisolated enantiomers, namely, (R)-BPO-27 and (S)-BPO-27. See, Example2A. As shown herein, while the (R)-BPO-27 enantiomer strongly inhibitedCFTR chloride conductance with IC₅₀-4 nM, the other enantiomer,(S)-BPO-27, was inactive.

CFTR inhibition potency was measured by short-circuit current analysisin FRT epithelial cells expressing human CFTR in presence of atransepithelial chloride gradient and in which the basolateral membranewas permeabilized with amphotericin B. Under these conditionsshort-circuit current is proportional to CFTR chloride conductance. FIG.10A shows no significant inhibition by BPO-27 fraction 1 at 100 nM,whereas BPO-27 fraction 2 at 100 nM completed inhibited current. FIG.10B shows the fraction 2 concentration-dependence, giving an IC₅₀ ofapproximately 4 nM, as compared to approximately 8 nM for racemic BPO-27(Example 31).

Example 34 In Vitro Metabolic Stability and Pharmacokinetics of IsolatedEnantiomers

The in vitro metabolic stability of (R)-BPO-27 and (S)-BPO-27 wasstudied using hepatic microsomes. Following compound incubation withhepatic microsomes in the presence of NADPH, remaining non-metabolizedcompound was assayed by LC/MS. FIG. 11A shows little metabolism ofeither BPO-27 isoform. As a control, PPQ-102 was >75% metabolized at 4 hwhen tested in parallel.

BPO-27 pharmacokinetics in mice was measured following bolusintraperitoneal administration in an aqueous formulation in 5% DMSO,2.5% Tween-80 and 2.5% PEG400 in water. BPO-27 was measured by LC/MS inkidney, blood and urine. FIG. 11B (left) shows ion current incalibration studies in which specified amounts of BPO-27 were added tokidney homogenates. The assay was linear with a detection sensitivity ofbetter than 100 nM BPO-27. FIG. 11B (right) shows the disappearancekinetics of BPO-27 from kidney following bolus intraperitonealadministration. FIG. 11C summarizes the deduced BPO-27 concentrations inkidney, blood and urine over time. The t_(1/2) for disappearance of theoriginal compound was approximately 2 h. Compound concentration remainedat predicted therapeutic levels (i.e., at levels much greater than 4 nM,which is the IC₅₀) for many hours following single dose administration.

The various embodiments described above can be combined to providefurther embodiments. All U.S. patents, U.S. patent applicationpublications, U.S. patent applications, foreign patents, foreign patentapplications, and non-patent publications referred to in thisspecification and/or listed in the Application Data Sheetareincorporated herein by reference, in their entirety. Aspects of theembodiments can be modified, if necessary, to employ concepts of thevarious patents, applications, and

These and other changes can be made to the embodiments in light of theabove-detailed publications to provide yet further embodiments.description. In general, in the following claims, the terms used shouldnot be construed to limit the claims to the specific embodimentsdisclosed in the specification and the claims, but should be construedto include all possible embodiments along with the full scope ofequivalents to which such claims are entitled. Accordingly, the claimsare not limited by the disclosure.

We claim the following:
 1. A compound having the following structure(I):

as an isolated enantiomer or a racemic mixture of enantiomers, or as apharmaceutically acceptable salt, hydrate, solvate, or N-oxide, thereof,wherein: m is 1, 2, 3, or 4; n is 1, 2, 3, 4 or 5; p is an integer from0 to 4; q is an integer from 1 to 4; R¹ at each occurrence is the sameor different and independently H, halo, haloalkyl, C₁-C₆ alkyl,—(CH₂)_(p)—C(O)—R^(4a), —S(O)₂R^(4a), —NO₂, or tetrazolyl; R^(1a) ateach occurrence is the same or different and independently H, halo,haloalkyl, C₁-C₆ alkyl, —(CH₂)_(p)—C(O)—R^(4a), —S(O)₂R^(4a), —NO₂, ortetrazolyl; R^(2a) and R^(2b) are each the same or different andindependently H, or C₁-C₆ alkyl; R^(4a) is —OR⁷, —NR⁷R⁸,—O(CH₂)_(q)—OC(O)R⁷, or an amino acid residue; R⁷ and R⁸ are each thesame or different and independently H, C₁-C₂₀ alkyl, a saccharide, or anamino acid residue; and Z is aryl or heteroaryl, wherein the amino acidresidue is selected from residues of alanine, arginine, asparagine,aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine,isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine,threonine, tryptophan, tyrosine, valine, phosphoserine,phosphothreonine, phosphotyrosine, 4-hydroxyproline, hydroxylysine,demosine, isodemosine desmosine, isodesmosine, gamma-carboxyglutamate,hippuric acid, octahydroindole-2-carboxylic acid, statine,1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, penicillamine,ornithine, 3-methylhistidine, norvaline, beta-alanine,gamma-aminobutylic gamma-aminobutyric acid, cirtulline citrulline,homocysteine, homoserine, methyl-alanine, para-benzoylphenylalanine,phenylglycine, propargylglycine, sarcosine, methionine sulfone,tert-butylglycine, 3,5-dibromotyrosine, 3,5-diiodotyrosine, glycosylatedthreonine, glyclosylated glycosylated serine, and glycosylatedasparagine.
 2. The compound of claim 1, wherein R^(2a) and R^(2b) areeach methyl, and Z is optionally substituted furanyl or optionallysubstituted thienyl, and the compound has the following structure (IA)or (IB), respectively:

as an isolated enantiomer or a racemic mixture of enantiomers, or as apharmaceutically acceptable salt, hydrate, solvate, or N-oxide thereof,wherein: m is 1, 2, 3, or 4; n is 1, 2, 3, 4 or 5; p is an integer from0 to 4; q is an integer from 1 to 4; R¹ at each occurrence is the sameor different and independently H, halo, haloalkyl, C₁₋₆ alkyl,—(CH₂)_(p)—C(O)—R^(4a), —S(O)₂R^(4a), —NO₂, or tetrazolyl; R^(1a) ateach occurrence is the same or different and independently H, halo,haloalkyl, C₁₋₆ alkyl, —(CH₂)_(p)—C(O)—R^(4a), —S(O)₂R^(4a), —NO₂, ortetrazolyl; R^(4a) is —OR⁷, —NR⁷R⁸, —O(CH₂)_(q)—OC(O)R⁷, or an aminoacid residue; R⁵ is H, halo, or C₁₋₆ alkyl; R⁶ is H, halo, C₁₋₆ alkyl,or C₁₋₆ haloalkyl; and R⁷ and R⁸ are each the same or different andindependently H, C₁₋₂₀ alkyl, a saccharide, or an amino acid residue,wherein the amino acid residue is selected from residues of alanine,arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid,glycine, histidine, isoleucine, leucine, lysine, methionine,phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine,phosphoserine, phosphothreonine, phosphotyrosine, 4-hydroxyproline,hydroxylysine, demosine, isodemosine desmosine, isodesmosine,gamma-carboxyglutamate, hippuric acid, octahydroindole-2-carboxylicacid, statine, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid,penicillamine, ornithine, 3-methylhistidine, norvaline, beta-alanine,gamma-aminobutylic gamma-aminobutyric acid, cirtulline citrulline,homocysteine, homoserine, methyl-alanine, para-benzoylphenylalanine,phenylglycine, propargylglycine, sarcosine, methionine sulfone,tert-butylglycine, 3,5-dibromotyrosine, 3,5-diiodotyrosine, glycosylatedthreonine, glyclosylated glycosylated serine, and glycosylatedasparagine.
 3. The compound of claim 1, wherein R^(2a) and R^(2b) areeach methyl, p is 0, R^(4a) is —OR⁷ is Z is optionally substitutedfuranyl, n is 1 and R¹ is meta to the linking carbon and the compoundhas the following structure (IA1):

wherein: R¹ is H, halo, or C₁₋₆ alkyl; R² and R³ are each the same ordifferent and independently H, halo, —NO₂, C₁₋₆ alkyl, tetrazolyl,—S(O)₂OR⁷, or —C(═O)OR⁷; R⁵ is H, halo, or C₁₋₆ alkyl; R⁶ is halo, C₁-C₆alkyl, or C₁₋₆ haloalkyl; and R⁷ is H, C₁₋₆ alkyl, a saccharide, or anamino acid residue, selected residues of alanine, arginine, asparagine,aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine,isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine,threonine, tryptophan, tyrosine, valine, phosphoserine,phosphothreonine, phosphotyrosine, 4-hydroxyproline, hydroxylysine,demosine, isodemosine desmosine, isodesmosine, gamma-carboxyglutamate,hippuric acid, octahydroindole-2-carboxylic acid, statine,1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, penicillamine,ornithine, 3-methylhistidine, norvaline, beta-alanine,gamma-aminobutylic gamma-aminobutyric acid, cirtulline citrulline,homocysteine, homoserine, methyl-alanine, para-benzoylphenylalanine,phenylglycine, propargylglycine, sarcosine, methionine sulfone,tert-butylglycine, 3,5-dibromotyrosine, 3,5-diiodotyrosine, glycosylatedthreonine, glyclosylated glycosylated serine, and glycosylatedasparagine.
 4. The compound of claim 1, wherein R^(2a) and R^(2b) areeach methyl, p is 0, R^(4a) is —OR⁷ , Z is optionally substitutedthienyl, n is 1 and R¹ is meta to the linking carbon, and the compoundhas the following structure (IB1):

wherein: R¹ is H, halo, or C₁₋₆ alkyl; R² and R³ are each the same ordifferent and independently H, halo, —NO₂, C₁₋₆ alkyl, tetrazolyl,—S(O)₂OR⁷, or —C(═O)OR⁷; R⁵ is H, halo, or C₁₋₆ alkyl; R⁶ is halo, C₁₋₆alkyl, or C₁₋₆ haloalkyl; and R⁷ is H, C₁₋₆ alkyl, a saccharide, or anamino acid residue selected from residues of alanine, arginine,asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine,histidine, isoleucine, leucine, lysine, methionine, phenylalanine,proline, serine, threonine, tryptophan, tyrosine, valine, phosphoserine,phosphothreonine, phosphotyrosine, 4-hydroxyproline, hydroxylysine,demosine, isodemosine desmosine, isodesmosine, gamma-carboxyglutamate,hippuric acid, octahydroindole-2-carboxylic acid, statine,1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, penicillamine,ornithine, 3-methylhistidine, norvaline, beta-alanine,gamma-aminobutylic gamma-aminobutyric acid, cirtulline citrulline,homocysteine, homoserine, methyl-alanine, para-benzoylphenylalanine,phenylglycine, propargylglycine, sarcosine, methionine sulfone,tert-butylglycine, 3,5-dibromotyrosine, 3,5-diiodotyrosine, glycosylatedthreonine, glyclosylated glycosylated serine, and glycosylatedasparagine.
 5. The compound of claim 1, wherein R^(2a) and R^(2b) areeach methyl, and Z is optionally substituted phenyl, and the compoundhas the following structure (IC):

as an isolated enantiomer or a racemic mixture of enantiomers, or as apharmaceutically acceptable salt, hydrate, solvate, or N-oxide thereof,wherein: m is 1, 2, 3, or 4; n is 1, 2, 3, 4 or 5; p is an integer from0 to 4; q is an integer from 1 to 4; t is 1, 2, 3, 4 or 5; R¹ at eachoccurrence is the same or different and independently H, halo,haloalkyl, C₁₋₆ alkyl, —(CH₂)_(p)—C(O)—R^(4a), —S(O)₂R^(4a), —NO₂, ortetrazolyl; R^(1a) at each occurrence is the same or different andindependently H, halo, haloalkyl, C₁₋₆ alkyl, —(CH₂)_(p)—C(O)—R^(4a),—S(O)₂R^(4a), —NO₂, or tetrazolyl; R^(1b) at each occurrence is the sameor different and independently H, halo, —OH, —NO₂, C₁₋₆ alkyl, C₂₋₆alkenyl, or C₁₋₆ alkoxy; R^(4a) is —OR⁷, —NR⁷R⁸, —O(CH₂)_(q)—OC(O)R⁷, oran amino acid residue; R⁷ and R⁸ are each the same or different andindependently H, C₁₋₂₀ alkyl, a saccharide, or an amino acid residue,wherein the amino acid residue is selected from residues of alanine,arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid,glycine, histidine, isoleucine, leucine, lysine, methionine,phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine,phosphoserine, phosphothreonine, phosphotyrosine, 4-hydroxyproline,hydroxylysine, demosine, isodemosine desmosine, isodesmosine,gamma-carboxyglutamate, hippuric acid, octahydroindole-2-carboxylicacid, statine, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid,penicillamine, ornithine, 3-methylhistidine, norvaline, beta-alanine,gamma-aminobutylic gamma-aminobutyric acid, cirtulline citrulline,homocysteine, homoserine, methyl-alanine, para-benzoylphenylalanine,phenylglycine, propargylglycine, sarcosine, methionine sulfone,tert-butylglycine, 3,5-dibromotyrosine, 3,5-diiodotyrosine, glycosylatedthreonine, glyclosylated glycosylated serine, and glycosylatedasparagine.
 6. The compound of claim 1, wherein R^(2a) and R^(2b) areeach methyl, p is 0, R^(4a) is —OR⁷, n is 1, and R¹ is meta to thelinking carbon and the compound has the following structure:

wherein: R¹ is H, halo, or C₁-C₆ alkyl; R² and R³ are each the same ordifferent and independently H, halo, —NO₂, C₁₋₆ alkyl, tetrazolyl,—S(O)₂OR⁷, or —C(═O)OR⁷; t is 1, 2, 3, 4 or 5; R^(1b) at each occurrenceis the same or different and independently H, halo, —OH, —NO₂, C₁₋₆alkoxy, or C₁₋₆ alkyl; and R⁷ is H, C₁₋₆ alkyl, a saccharide, or anamino acid residue selected from residues of alanine, arginine,asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine,histidine, isoleucine, leucine, lysine, methionine, phenylalanine,proline, serine, threonine, tryptophan, tyrosine, valine, phosphoserine,phosphothreonine, phosphotyrosine, 4-hydroxyproline, hydroxylysine,demosine, isodemosine desmosine, isodesmosine, gamma-carboxyglutamate,hippuric acid, octahydroindole-2-carboxylic acid, statine,1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, penicillamine,ornithine, 3-methylhistidine, norvaline, beta-alanine,gamma-aminobutylic gamma-aminobutyric acid, cirtulline citrulline,homocysteine, homoserine, methyl-alanine, para-benzoylphenylalanine,phenylglycine, propargylglycine, sarcosine, methionine sulfone,tert-butylglycine, 3,5-dibromotyrosine, 3,5-diiodotyrosine, glycosylatedthreonine, glyclosylated glycosylated serine, and glycosylatedasparagine.
 7. A compound having the following structure (II):

as an isolated enantiomer or a racemic mixture of enantiomers, or as apharmaceutically acceptable salt, hydrate, solvate, or N-oxide thereof,wherein: m is 1, 2, 3, or 4; n is 1, 2, 3, 4 or 5; p is an integer from0 to 4; q is an integer from 1 to 4; X is O or S; R¹ at each occurrenceis the same or different and independently H, halo, haloalkyl, C₁₋₆alkyl, —(CH₂)_(p)—C(O)—R^(4a), —S(O)₂R^(4a), —NO₂, or tetrazolyl; R^(1a)at each occurrence is the same or different and independently H, halo,haloalkyl, C₁₋₆ alkyl, —(CH₂)_(p)—C(O)—R^(4a), —S(O)₂R^(4a), —NO₂, ortetrazolyl; R^(2a) and R^(2b) are each the same or different andindependently H or C₁₋₆ alkyl; R^(4a) is —OR⁷, —NR⁷R⁸,—O(CH₂)_(q)—OC(O)R⁷, an amino acid residue; R⁴ is H, —N(═O), C₁₋₆ alkyl,or haloalkyl; R⁵ is H, halo, or C₁₋₆ alkyl; R⁶ is halo; and R⁷ and R⁸are each the same or different and independently H, C₁-C₂₀ alkyl, asaccharide, or an amino acid residue; wherein the amino acid residue isselected from residues of alanine, arginine, asparagine, aspartic acid,cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine,leucine, lysine, methionine, phenylalanine, proline, serine, threonine,tryptophan, tyrosine, valine, phosphoserine, phosphothreonine,phosphotyrosine, 4-hydroxyproline, hydroxylysine, demosine, isodemosinedesmosine, isodesmosine, gamma-carboxyglutamate, hippuric acid,octahydroindole-2-carboxylic acid, statine,1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, penicillamine,ornithine, 3-methylhistidine, norvaline, beta-alanine,gamma-aminobutylic acid gamma-aminobutyric, cirtulline citrulline,homocysteine, homoserine, methyl-alanine, para-benzoylphenylalanine,phenylglycine, propargylglycine, sarcosine, methionine sulfone,tert-butylglycine, 3,5-dibromotyrosine, 3,5-diiodotyrosine, glycosylatedthreonine, glyclosylated glycosylated serine, and glycosylatedasparagine.
 8. The compound of claim 7, wherein R^(2a) and R^(2b) areeach methyl, p is 0, R^(4a) is —OR⁷, n is 1 and R¹ is meta to thelinking carbon, and the compound has the following structure (IIA):

wherein: X is O or S; R¹ is H, halo, or C₁₋₃ alkyl; R² is H, halo, —NO₂,or —C(═O)OR⁷; R³ is H or —NO₂; R⁴ is —N(═O), C₁₋₃ alkyl, or H; R⁵ is Hor C₁₋₃ alkyl; R⁶ halo; and R⁷ is H, C₁₋₆ alkyl, a saccharide, an aminoacid residue of alanine, arginine, asparagine, aspartic acid, cysteine,glutamine, glutamic acid, glycine, histidine, isoleucine, leucine,lysine, methionine, phenylalanine, proline, serine, threonine,tryptophan, tyrosine, valine, phosphoserine, phosphothreonine,phosphotyrosine, 4-hydroxyproline, hydroxylysine, demosine, isodemosinedesmosine, isodesmosine, gamma-carboxyglutamate, hippuric acid,octahydroindole-2-carboxylic acid, statine,1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, penicillamine,ornithine, 3-methylhistidine, norvaline, beta-alanine,gamma-aminobutylic gamma-aminobutyric acid, cirtulline citrulline,homocysteine, homoserine, methyl-alanine, para-benzoylphenylalanine,phenylglycine, proparagylglycine, sarcosine, methionine sulfone,tert-butylglycine, 3,5-dibromotyrosine, 3,5-diiodotyrosine, glycosylatedthreonine, glyclosylated glycosylated serine, and glycosylatedasparagine.
 9. The compound of claim 1, wherein (a) each R¹ is H; (b) atleast one R¹ is methyl or ethyl; (c) at least one R¹ is methyl and eachremaining R¹ is H; or (d) at least one R¹ is ethyl and each remaining R¹is H.
 10. The compound of claim 1, wherein m=2 and p=0 and (a) at leastone of R^(1a) is halo, —NO₂, C₁₋₃ alkyl, —C(O)—R^(4a); or (b) each ofR^(1a) is H.
 11. The compound of claim 5, wherein (a) each R¹ is H; (b)at least one R¹ is methyl or ethyl; (c) at least one R¹ is methyl andeach remaining R¹ is H; or (d) at least one R¹ is ethyl and eachremaining R¹ is H.
 12. The compound of claim 5, wherein m=2 and p=0 and(a) at least one of R^(1a) is halo, —NO₂, C₁₋₃ alkyl, —C(O)—R^(4a); or(b) each of R^(1a) is H.
 13. The compound of claim 6, wherein (a) R² isH, halo, —NO₂, or —C(═O)OR⁷, wherein R⁷ is H or C₁-C₆ alkyl; (b) R² isH, halo, —NO₂, or —C(═O)OR⁷, wherein R⁷ is H, —CH₃, —CH₂CH₃, or—CH₂CH₂CH₃; (c) R² is H, chloro, or —NO₂; (d) R² is —C(═O)OR⁷, whereinR⁷ is H, —CH₃, or —CH₂CH₃; or (e) R² is chloro.
 14. The compound ofclaim 5, wherein: (a) t=2 and each R^(1b) is the same or different andindependently H, —OH, halo, —NO₂, C₁₋₃ alkyl, or C₁₋₃ alkoxy; or (b) t=1and R^(1b) is —OH, halo, —NO₂, C₁₋₃ alkyl, or C₁₋₃ alkoxy; or (c) t=2and each R^(1b) is the same or different and independtly independentlyH, —OH, chloro, fluoro, —NO₂, methyl or methoxy; or (d) t=1 and R^(1b)is —OH, chloro, flouro fluoro, —NO₂, methyl, or methooxy; (c)(e) eachR^(1b) is H.
 15. The compound of claim 3 wherein (a) R² is H, halo,—NO₂, or —C(═O)OR⁷, wherein R⁷ is H or C₁₋₆ alkyl; (b) R² is H, halo,—NO₂, or —C(═O)OR⁷, wherein R⁷ is H, —CH₃, —CH₂CH₃, or —CH₂CH₂CH₃; (c)R² is H, chloro, or —NO₂; (d) R² is —C(═O)OR⁷, wherein R⁷ is H, —CH₃, or—CH₂CH₃; or (e) R² is chloro.
 16. The compound of claim 4 wherein (a) R²is H, halo, —NO₂, or —C(═O)OR⁷, wherein R⁷ is H or C₁₋₆ alkyl; (b) R² isH, halo, —NO₂, or —C(═O)OR⁷, wherein R⁷ is H, —CH₃, —CH₂CH₃, or—CH₂CH₂CH₃; (c) R² is H, chloro, or —NO₂; (d) R² is —C(═O)OR⁷, whereinR⁷ is H, —CH₃, or —CH₂CH₃; or (e) R² is chloro.
 17. The compound ofclaim 2, wherein (a) each R¹ is H; (b) at least one R¹ is methyl orethyl; (c) at least one R¹ is methyl and each remaining R¹ is H; or (d)at least one R¹ is ethyl and each remaining R¹ is H.
 18. The compound ofclaim 7, wherein (a) each R¹ is H; (b) at least one R¹ is methyl orethyl; (c) at least one R¹ is methyl and each remaining R¹ is H; or (d)at least one R¹ is ethyl and each remaining R¹ is H.
 19. The compound ofclaim 2, wherein m=2 and p=0 and (a) at least one of R^(1a) is halo,—NO₂, C₁-C₃ alkyl, —C(O)—R^(4a); or (b) each of R^(1a) is H.
 20. Thecompound of claim 7, wherein m=2 and p=0 and (a) at least one of R^(1a)is halo, —NO₂, C₁₋₃ alkyl, —C(O)—R^(4a); or (b) each of R^(1a) is H. 21.The compound of claim 2, wherein R⁶ is chloro, bromo, or iodo.
 22. Thecompound of claim 1, wherein the compound has any one of the followingstructures:


23. The compound of claim 7, wherein the compound has any one of thefollowing structures:


24. The compound of claim 1, wherein the compound is an isolatedenantiomer in R form.
 25. The compound of claim 24 wherein the compoundis6R-(5-Bromofuran-2-yl)-7,9-dimethyl-8,10-dioxo-11-phenyl-7,8,9,10-tetrahydro-6H-benzo[b]pyrimido[4′,5′:3,4]pyrrolo [1,2-d][1,4]oxazine-2-carboxylic acid. 26.The compound of claim 1, wherein the compound is an isolated enantiomerin S form.
 27. A pharmaceutical composition comprising the compound ofclaim 1 and a pharmaceutically suitable excipient.
 28. A method forinhibiting cyst formation or inhibiting cyst enlargement, said methodcomprising contacting (a) a cell that comprises CFTR and (b) thepharmaceutical composition of claim 27, under conditions and for a timesufficient that permit CFTR and the compound to interact, wherein thecompound inhibits CFTR-mediated ion transport.
 29. A method for treatinga disease, condition, or disorder that is treatable by inhibiting cysticfibrosis transmembrane conductance regulator (CFTR)-mediated iontransport, said method comprising administering to a subject thepharmaceutical composition of claim 27, thereby inhibiting CFTR-mediatedion transport.
 30. The method of claim 29, wherein the disease,condition, or disorder is selected from polycystic kidney disease,aberrantly increased intestinal fluid secretion, and secretory diarrhea.31. A pharmaceutical composition comprising the compound of claim 7 anda pharmaceutically suitable excipient.
 32. A method for inhibiting cystformation or inhibiting cyst enlargement, said method comprisingcontacting (a) a cell that comprises CFTR and (b) the pharmaceuticalcomposition of claim 31, under conditions and for a time sufficient thatpermit CFTR and the compound to interact, wherein the compound inhibitsCFTR-mediated ion transport.
 33. A method for treating a disease,condition, or disorder that is treatable by inhibiting cystic fibrosistransmembrane conductance regulator (CFTR)-mediated ion transport, saidmethod comprising administering to a subject the pharmaceuticalcomposition of claim 31, thereby inhibiting CFTR-mediated ion transport.34. The method of claim 33, wherein the disease, condition, or disorderis selected from polycystic kidney disease, aberrantly increasedintestinal fluid secretion, and secretory diarrhea.
 35. The compound ofclaim 22, wherein said compound has the structure:


36. The compound of claim 1, wherein the compound is a racemic mixtureof enantiomers.
 37. The compound of claim 36, which is a racemic mixtureof6R-(5-Bromofuran-2-yl)-7,9-dimethyl-8,10-dioxo-11-phenyl-7,8,9,10-tetrahydro-6H-benzo[b]pyrimido[4′,5′:3,4]pyrrolo[1,2-d][1,4]oxazine-2-carboxylicacid; and6S-(5-Bromofuran-2-yl)-7,9-dimethyl-8,10-dioxo-11-phenyl-7,8,9,10-tetrahydro-6H-benzo[b]pyrimido[4′,5′:3,4]pyrrolo[1,2-d][1,4]oxazine-2-carboxylicacid.
 38. A pharmaceutical composition comprising the compound of claim35 and a pharmaceutically suitable excipient.
 39. A pharmaceuticalcomposition comprising the compound of claim 37 and a pharmaceuticallysuitable excipient.
 40. A method for inhibiting cyst formation orinhibiting cyst enlargement, said method comprising contacting (a) acell that comprises CFTR and (b) the pharmaceutical composition of claim38, under conditions and for a time sufficient that permit CFTR and thecompound to interact, wherein the compound inhibits CFTR-mediated iontransport.
 41. A method for treating a disease, condition, or disorderthat is treatable by inhibiting cystic fibrosis transmembraneconductance regulator (CFTR)-mediated ion transport, said methodcomprising administering to a subject the pharmaceutical composition ofclaim 38, thereby inhibiting CFTR-mediated ion transport.
 42. The methodof claim 41, wherein the disease, condition, or disorder is selectedfrom polycystic kidney disease, aberrantly increased intestinal fluidsecretion, and secretory diarrhea.
 43. A method for inhibiting cystformation or inhibiting cyst enlargement, said method comprisingcontacting (a) a cell that comprises CFTR and (b) the pharmaceuticalcomposition of claim 39, under conditions and for a time sufficient thatpermit CFTR and the compound to interact, wherein the compound inhibitsCFTR-mediated ion transport.
 44. A method for treating a disease,condition, or disorder that is treatable by inhibiting cystic fibrosistransmembrane conductance regulator (CFTR)-mediated ion transport, saidmethod comprising administering to a subject the pharmaceuticalcomposition of claim 39, thereby inhibiting CFTR-mediated ion transport.45. The method of claim 44, wherein the disease, condition, or disorderis selected from polycystic kidney disease, aberrantly increasedintestinal fluid secretion, and secretory diarrhea.
 46. The method ofclaim 44, wherein the disease, condition, or disorder is polycystickidney disease.
 47. The method of claim 44, wherein the disease,condition, or disorder is aberrantly increased intestinal fluidsecretion.
 48. The method of claim 44, wherein the disease, condition,or disorder is secretory diarrhea.
 49. A method for treating polycystickidney disease, aberrantly increased intestinal fluid secretion, orsecretory diarrhea in a subject in need thereof, the method comprisingadministering to the subject a racemic mixture of6R-(5-Bromofuran-2-yl)-7,9-dimethyl-8,10-dioxo-11-phenyl-7,8,9,10-tetrahydro-6H-benzo[b]pyrimido[4′,5′:3,4]pyrrolo[1,2-d][1,4]oxazine-2-carboxylicacid; and6S-(5-Bromofuran-2-yl)-7,9-dimethyl-8,10-dioxo-11-phenyl-7,8,9,10-tetrahydro-6H-benzo[b]pyrimido[4′,5′:3,4]pyrrolo[1,2-d][1,4]oxazine-2-carboxylicacid.
 50. The method of claim 49, for the treatment of polycystic kidneydisease.
 51. The method of claim 49, for the treatment of aberrantlyincreased intestinal fluid secretion.
 52. The method of claim 49, forthe treatment of secretory diarrhea.